JP3590585B2 - Antisense regulation of LFA-3 - Google Patents

Abstract

Compositions and methods for the treatment and diagnosis of diseases or disorders amenable to treatment through modulation of expression of a nucleic acid encoding a lymphocyte function associated antigen 3 (LFA-3; also known as CD58) protein are provided.

Description

【０１３０】
実施例３：ＨＵＶＥＣ細胞におけるＬＦＡ−３発現のアンチセンス介在阻害の検定
Ａ．一般的な技術：潜在的なヒトＬＦＡ−３調節性オリゴヌクレオチドの活性を評価するため、Ｃｌｏｎｅｔｉｃｓ Ｃｏｒｐｏｒａｔｉｏｎ （Ｗａｌｋｅｒｓｖｉｌｌｅ、ＭＤ）からのヒト臍静脈内皮細胞（ＨＵＶＥＣ）（その代わりに、Ａｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ、Ｒｏｃｋｖｉｌｌｅ 、ＭＤからのＡＴＣＣ ＣＲＬ−１７３０を用いることができる）を成長させ、オリゴヌクレオチド又は対照溶液で下記のように処理した。回収後、培養抽出物を調製し、ＬＦＡ−３ ＲＮＡ及びタンパク質濃度について調べた（例えば、それぞれ、ノーザン又はフローサイトメトリー検定）。全ての場合において、“発現％” は非処理細胞（又はオリゴヌクレオチドを欠く対照溶液で処理した細胞）に対するオリゴヌクレオチド処理細胞におけるＬＦＡ−３特異的シグナルの量を指し、“阻害％” は以下のように算出する：
１００％ − 発現％ ＝ 阻害％
【０１３１】
Ｂ．ＲＮＡ検定：各ＬＦＡ−３タンパク質のｍＲＮＡの発現を、それらに特異的にハイブリダイズし得る核酸プローブを用いて“ノーザン” 検定で決定した。ＬＦＡ−３に特異的な核酸プローブはこれらの例に記載されている。これらのプローブは当該技術分野において公知の手段で放射標識した（例えば、Ｓｈｏｒｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， ２ｎｄ Ｅｄ．， Ａｕｓｕｂｅｌ ｅｔ ａｌ．， ｅｄｓ．， Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ， Ｎｅｗ Ｙｏｒｋ， １９９２， ｐａｇｅｓ ３−１１〜２−３−４４及び ４−１７ 〜 ４−１８ ；Ｍｅｔｈｏｄｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， Ｖｏｌ． ２６： Ｐｒｏｔｏｃｏｌｓ ｆｏｒ Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ Ｃｏｎｊｕｇａｔｅｓ， Ａｇｒａｗａｌ， ｅｄ．， Ｈｕｍａｎａ Ｐｒｅｓｓ Ｉｎｃ．， Ｔｏｔｏｗａ， ＮＪ， １９９４， ｐａｇｅｓ １６７−１８５ の Ｒｕｔｈ 、６ 章；及び Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ， ２ｎｄ Ｅｄ．， Ｓａｍｂｒｏｏｋ ｅｔ ａｌ．， ｅｄｓ．， ｐａｇｅｓ １０．１−１０．７０ の １０ 章を参照）。ＲＮＡのロードが等しいことを確認し、かつＬＦＡ−３転写体の濃度をＧ３ＰＤＨシグナルに関して標準化するため、ブロットを細片にし、^３２Ｐ標識グリセルアルデヒド３−ホスフェートデヒドロゲナーゼ（Ｇ３ＰＤＨ）プローブ（Ｃｌｏｎｔｅｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ， Ｉｎｃ． 、Ｐａｌｏ Ａｌｔｏ 、ＣＡ）を用いて再検索した。
【０１３２】
ＨＵＶＥＣ（ヒト）細胞を、Ｔ−７５フラスコ内で、８０〜９０％集密まで、１０％ウシ胎児血清（ＦＢＳ）を含むＥＧＭ（Ｃｌｏｎｅｔｉｃｓ 、Ｗａｌｋｅｒｓｖｉｌｌｅ、ＭＤ）培地中で成長させた。この時点で、細胞を１０ｍＬのＯｐｔｉ−ＭＥＭ培地（ＧＩＢＣＯ−ＢＲＬ ）で３回洗浄した。次に、１０μ／ｍＬ ＬＩＰＯＦＥＣＴＩＮ（登録商標）（すなわち、１：１（ｗ／ｗ）ＤＯＴＭＡ／ＤＯＰＥ、ここで、ＤＯＴＭＡ＝Ｎ−［１−（２，３−ジオレイオキシ）プロピル］−Ｎ，Ｎ，Ｎ−トリメチルアンモニウム塩化物であり、かつＤＯＰＥ＝ジオレオイルホスファチジルエタノールアミン；ＧＩＢＣＯ−ＢＲＬ ）を含む５ｍＬのＯｐｔｉ−ＭＥＭ培地及び適量のオリゴヌクレオチドを細胞に添加した（ここに記載される実験においてオリゴヌクレオチドの添加時間はｔ＝０ｈである）。対照として、細胞を、オリゴヌクレオチド処理試料と同じ条件下で、同じ時間、オリゴヌクレオチドを含まないＬＩＰＯＦＥＣＴＩＮ（登録商標）で処理した。３７℃で４時間後（ｔ＝４ｈ）、培地を、１０％ＦＢＳを含む新鮮なＥＧＭ培地で置き換えた。細胞を典型的には２時間回復させた。次に、当該技術分野において公知の技術に従い、全細胞性ＲＮＡをグアニジニウム中で抽出し、ゲル電気泳動にかけてフィルターに移した（例えば、Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ， ２ｎｄ Ｅｄ．， Ｓａｍｂｒｏｏｋ ｅｔ ａｌ．， ｅｄｓ．， ｐａｇｅｓ ７．１−７．８７の ７章、及び Ｓｈｏｒｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， ２ｎｄ Ｅｄ．， Ａｕｓｕｂｅｌ ｅｔ ａｌ．， ｅｄｓ．， Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ， Ｎｅｗ Ｙｏｒｋ， １９９２， ｐａｇｅｓ ２−２４ ｔｏ ２−３０ ａｎｄ ４−１４ ｔｏ ４−２９を参照）。
【０１３３】
ＲＮＡ転移に続いて、フィルターを、関心のある特定のＬＦＡ−３コード化遺伝子に対して特異的なプローブと、ハイブリダイゼーションバッファ（ＱＵＩＫＨＹＢ（商標）ハイブリダイゼーション溶液、Ｓｔｒａｔａｇｅｎｅ、Ｌａ Ｊｏｌｌａ、ＣＡ）中で、典型的には一晩ハイブリダイズさせる。これに続いて、室温（〜２４℃）で１５分間、２×ＳＳＣ、０．１％ＳＤＳで２回、及び６０℃で３０分間、０．１×ＳＳＣ、０．１％ＳＤＳで１回洗浄した。ハイブリダイズするバンドをＸ−ＯＭＡＴ ＡＲフィルムに露光することによって可視化し、ＰＨＯＳＰＨＯＲＩＭＡＧＥＲ（登録商標）を用いて、本質的には製造者の指示（Ｍｏｌｅｃｕｌａｒ Ｄｙｎａｍｉｃｓ、Ｓｕｎｎｙｖａｌｅ 、ＣＡ）に従って定量した。ＰＨＯＳＰＨＯＲＩＭＡＧＥＲ（登録商標）又は匹敵する器機による定量化がＲＮＡ濃度測定の好ましい手段ではあるが、これらの“ノーザン” 検定の結果は当該技術分野において公知の他の手段によって決定することができる。
【０１３４】
Ｃ．タンパク質検定：ＨＵＶＥＣ細胞を、１２ウェルプレートフラスコ内で、８０〜９０％集密まで、１０％ＦＢＳを含むＥＧＭ（Ｃｌｏｎｅｔｉｃｓ 、Ｗａｌｋｅｒｓｖｉｌｌｅ、ＭＤ）培地中で成長させた。この時点で、細胞を１０ｍＬのＯｐｔｉ−ＭＥＭ培地（ＧＩＢＣＯ−ＢＲＬ ）で３回洗浄した。次に、１０μｇ／ｍＬ ＬＩＰＯＦＥＣＴＩＮ（登録商標）［すなわち、１：１（ｗ／ｗ）ＤＯＴＭＡ／ＤＯＰＥ、ここで、ＤＯＴＭＡ＝Ｎ−［１−（２，３−ジオレイオキシ）プロピル］−Ｎ，Ｎ，Ｎ−トリメチルアンモニウム塩化物であり、ＤＯＰＥ＝ジオレオイルホスファチジルエタノールアミンである；ＧＩＢＣＯ−ＢＲＬ ）を含む５００μＬのＯｐｔｉ−ＭＥＭ培地及び適量のオリゴヌクレオチドを細胞に添加した（ここに記載される実験においてオリゴヌクレオチドの添加時間はｔ＝０ｈである）。対照として、オリゴヌクレオチド処理試料と同じ条件下で同じ時間、オリゴヌクレオチドを含まないＬＩＰＯＦＥＣＴＩＮ（登録商標）で処理した。３７℃で４時間後（ｔ＝４ｈ）、培地を、１０％ＦＢＳを含む新鮮なＥＧＭ培地で置き換えた。タンパク質抽出物の調製に先立ち、細胞を典型的には４８時間回復させた。細胞を洗浄し、トリプシン（ＧＩＢＣＯ−ＢＲＬ ）で処理することによって放出させた。細胞を、検査中のＬＦＡ−３タンパク質を特異的に認識する、適切に蛍光標識した一次抗体で染色し、蛍光活性化細胞ソーティング（ＦＡＣＳ（登録商標））技術（例えば、Ｒｅｃｋｔｅｎｗａｌｄ の米国特許第 ４，７２７，０２０号、Ｋｏｒｔｒｉｇｈｔ らの第 ５，２２３，３９８号及び Ｓｉｚｔｏらの第 ５，５５６，７６４号を参照）を用いて各ＬＦＡ−３タンパク質の量を決定した。各ＬＦＡ−３タンパク質に特異的な蛍光標識一次抗体は適切な実施例に記載されている。その代わりに、ＬＦＡ−３に対する非標識一次抗体（幾つかがこれらの実施例に記載されている）を用い、蛍光標識二次 （すなわち、一次抗体に特異的な）抗体を用いることによって検出することができる。
【０１３５】
ＬＦＡ−３濃度を測定するための代替法においては、細胞溶解物及びタンパク質抽出物を電気泳動し（ＳＤＳ−ＰＡＧＥ）、ニトロセルロースフィルターに移して当該技術分野において公知の手段によって検出する（例えば、Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ， ２ｎｄ Ｅｄ．， Ｓａｍｂｒｏｏｋ ｅｔ ａｌ．， ｅｄｓ．， ｐａｇｅｓ １８．３４， １８．４７−１８．５４ ａｎｄ １８．６０−１８．７５の １８ 章を参照）。ＬＦＡ−３に対する非標識一次抗体を用い、例えば、その一次抗体に結合する二次抗体による一次抗体の検出を含む、当該技術分野において公知の手段によって検出し（例えば、Ｓｈｏｒｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， ２ｎｄ Ｅｄ．， Ａｕｓｕｂｅｌ ｅｔ ａｌ．， ｅｄｓ．， Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ， Ｎｅｗ Ｙｏｒｋ， １９９２， ｐａｇｅｓ １０−３３ ｔｏ １０−３５；及び Ｍｏｌｅｃｕｌａｒ Ｃｌｏｎｉｎｇ： Ａ Ｌａｂｏｒａｔｏｒｙ Ｍａｎｕａｌ， ２ｎｄ Ｅｄ．， Ｓａｍｂｒｏｏｋ ｅｔ ａｌ．， ｅｄｓ．， ｐａｇｅｓ １８．１−１８．７５ ａｎｄ １８．８６−１８．８８ の １８ 章を参照）、かつ当該技術分野において公知の他の抗体ベースの検定を用いて定量することができる。このような抗体ベースの検定には、ＥＬＩＳＡ検定、ウェスタン検定等が含まれるが、これらに限定されるものではない（例えば、Ｗａｎｄｓ らの米国特許第 ４，８７９，２１９号及び Ｓｃｈｏｅｍａｋｅｒ らの第 ４，８３７，１６７号並びに Ｓｈｏｒｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ， ２ｎｄ Ｅｄ．， Ａｕｓｕｂｅｌ ｅｔ ａｌ．， ｅｄｓ．， Ｊｏｈｎ Ｗｉｌｅｙ ＆ Ｓｏｎｓ， Ｎｅｗ Ｙｏｒｋ， １９９２， ｐａｇｅｓ １１−５ ｔｏ １１−１７を参照）。ＬＦＡ−３の活性は当該技術分野において公知の適切な細胞接着検定及びモデル系において測定することができる（Ｄｕｓｔｉｎ ｅｔ ａｌ．， Ａｎｎｕ． Ｒｅｖ． Ｉｍｍｕｎｏｌ．， １９９１， ９， ２７ ）。
【０１３６】
実施例４：オリゴヌクレオチドによるヒトＬＦＡ−３の発現のアンチセンス介在阻害
Ａ．ＬＦＡ−３オリゴヌクレオチドの活性：ＬＦＡ−３の発現を調節することができる活性化合物の初期スクリーニングにおいて、ＨＵＶＥＣ細胞を１０ｎＭの一連のＬＦＡ−３特異的“ギャップマー” オリゴヌクレオチド（ＩＳＩＳ番号１６３７１〜１６３８１、１６９０９〜１６９１２、１７０９２及び１７１５９；配列番号２〜１５）で処理し、ＬＦＡ−３タンパク質の濃度を、ＦＡＣＳ（登録商標）により、Ｐｈａｒｍｉｎｇｅｎ（Ｓａｎ Ｄｉｅｇｏ 、ＣＡ）からのフィコエリスリン（ＰＥ）に結合した、ＬＦＡ−３（ＣＤ５８）に対するモノクローナル抗体を用いて決定した。アイソタイプ（マウスＩｇＧ−２ａカッパ）対照抗体（同様に Ｐｈａｒｍｉｎｇｅｎ から）は、典型的には、基本シグナルの≦５％のシグナルをもたらした。ＬＦＡ−３に対するＰＥ−及びＦＩＴＣ−標識抗体は、例えば、Ｉｍｍｕｎｏｔｅｃｈ（Ｗｅｓｔｂｒｏｏｋ 、ＭＥ）、Ｓｅｒｏｔｅｃ （Ｏｘｆｏｒｄ、英国）、Ｂｉｏｄｅｓｉｇｎ Ｉｎｔｅｒｎａｔｉｏｎａｌ （Ｋｅｎｎｅｂｕｎｋ 、ＭＥ）、Ｒｅｓｅａｒｃｈ Ｄｉａｇｎｏｓｔｉｃｓ， Ｉｎｃ．（ＲＤＩ 、Ｆｌａｎｄｅｒｓ、ＮＪ）から入手可能である。標識二次抗体との組み合わせでＦＡＣＳ（登録商標）分析に適する、他のＬＦＡ−３に対する一次抗体は、上記ベンダー及び、例えば、Ｅｎｄｏｇｅｎ （かつては Ｔ Ｃｅｌｌ Ｄｉａｇｎｏｓｔｉｃｓ 、Ｗｏｂｕｒｎ、ＭＡ）及び Ｕｐｓｔａｔｅ Ｂｉｏｔｅｃｈｎｏｌｏｇｙ（Ｌａｋｅ Ｐｌａｃｉｄ 、ＮＹ）から入手可能である。
【０１３７】
スクリーニングからのデータ（表２）は以下の結果を示す。この検定において、阻害レベル≧ＬＦＡ−３タンパク質濃度の約３０％（ここで、“約” は±５％を示す）に反映される活性を示すオリゴヌクレオチドには、ＩＳＩＳ番号１６３７１、１６３７３、１６３７４、１６３７６、１６３７７、１６３７８、１６９０９、１６９１０及び１７１５９（それぞれ、配列番号２、４、５、７、８、９、５、２、及び５）が含まれる。したがって、これらのオリゴヌクレオチドは、ＬＦＡ−３の発現を調節するための本発明の好ましい態様である。この検定において阻害レベル≧ＬＦＡ−３の約５０％（ここで、“約” は±５％を示す）を示すオリゴヌクレオチドには、ＩＳＩＳ番号１６３７１、１６３７４、１６９１０及び１７１５９（それぞれ、配列番号２、５、２及び５）が含まれる。したがって、これらのオリゴヌクレオチドはＬＦＡ−３の発現を調節するための本発明の最も好ましい態様である。
【０１３８】
【表２】

【０１３９】
Ｂ．用量応答及び配列特異性：様々な濃度のＩＳＩＳ番号１６３７１、１６３７４、１６３７５及び１３３１５での処理の４時間後の、ＨＵＶＥＣ細胞におけるＬＦＡ−３タンパク質の発現の用量応答が表３に示されている。ＩＳＩＳ１３３１５（配列番号１６）はマウスＩＣＡＭ−１を標的とするアンチセンス・オリゴヌクレオチドであり、無関連配列対照として用いた。ＬＦＡ−３タンパク質の濃度は上述の通りＦＡＣＳ（登録商標）によって決定した。これらの条件下で、活性アンチセンス・オリゴヌクレオチドＩＳＩＳ１６３７１及びＩＳＩＳ１６３７４は、１０ｎＭの濃度で適用したとき、それぞれ、ＬＦＡ−３タンパク質濃度の約４５％及び７０％阻害をもたらし、ここで“約” は±５％を示す。対照的に、不活性アンチセンス・オリゴヌクレオチドＩＳＩＳ１６３７５では最小阻害、すなわち、≦約１５％阻害がもたらされた。ＩＳＩＳ１６３７１については、阻害の程度は３０ｎＭで約６５％に増加した。ＩＳＩＳ１６３７４については、阻害の程度は３０ｎＭで約８０％に増加した。
【０１４０】
アンチセンス・オリゴヌクレオチドによるＬＦＡ−３の調節の特異性を、ＩＳＩＳ番号１３３１５を対照として用いて確認した。それらの結果は、対照オリゴヌクレオチドでの細胞の処理がＬＦＡ−３タンパク質の発現に対してほとんど効果がないことを示す。これらの結果は、スクランブルされた対照オリゴヌクレオチドＩＳＩＳ番号１６９１１、１６９１２、１７０９２について表２に示される結果と一致する。これらのスクランブルされた対照オリゴヌクレオチドはＬＦＡ−３の最小阻害、すなわち、≦約１０％（ここで、“約” は±５％を示す）をももたらした。これらの結果は、活性アンチセンス化合物のＬＦＡ−３調節活性が配列特異的であることを示す。
【０１４１】
【表３】

【０１４２】
Ｃ．アンチセンス機構の確認：活性と同定されたオリゴヌクレオチドがアンチセンス機構で作用することを確認するため、以下の実験を行ってＬＦＡ−３ｍＲＮＡの濃度を検定した。ＬＦＡ−３特異的核酸プローブを調製するため、ＰＲＩＭＥ−Ａ−ＧＥＮＥ（登録商標）標識キット（Ｐｒｏｍｅｇａ 、Ｍａｄｉｓｏｎ 、ＷＩ）を用いるＰＣＲ反応において、ＨＵＶＥＣ細胞に由来するＲＮＡ及び一対のホスホジエステルオリゴヌクレオチド、
５’−ＣＡＧＣＧＴＧＧＴＣＴＧＣＣＴＧＣＴＧＣ （配列番号１７）；及び
５’−ＧＴＴＡＴＧＣＴＧＴＴＧＴＣＴＴＣＡＴＣ （配列番号１８）、
をプライマーとして用いて、放射標識ＬＦＡ−３特異的ＰＣＲ産物を調製した。得られた^３２Ｐ−シトシン放射標識７４３ｂｐ産物はヒトＬＦＡ−３の読み取り枠（配列番号１）にハイブリダイズする。しかしながら、その代わりに、他のＰＣＲプローブを調製することができ、又は当該技術分野において公知の方法を用いて表１のオリゴヌクレオチドの１つ以上を検出可能に標識してＬＦＡ−３特異的プローブとして用いることができる。
【０１４３】
ノーザン検定は、上記プローブを用いて、実施例３に記述される通りに行った。ＨＵＶＥＣ細胞を５ｎＭのアンチセンス・オリゴヌクレオチドで処理し、ｔ＝ｈにＲＮＡを回収した。この実験を３回行い、それらの結果をＰＨＯＳＰＨＯＲＩＭＡＧＥＲ（登録商標）を用いて定量し、グリセルアルデヒド３−ホスフェートデヒドロゲナーゼ（Ｇ３ＰＤＨ）をコードするｍＲＮＡに対して標準化した。これらの結果（表４）は、ＩＳＩＳ１６３７４がＬＦＡ−３ ｍＲＮＡの濃度をほぼ８５％減少させ、これに対して対照（スクランブルされた）オリゴヌクレオチドはＬＦＡ−３ ｍＲＮＡの濃度に対する効果がほとんどないことを示す。したがって、本発明の化合物はそれらの効果を紛うことなくアンチセンス機構で発揮する。
【０１４４】
【表４】

【０１４５】
Ｄ．ヒトＬＦＡ−３をコードする核酸のアンチセンス調節に好ましい領域：本発明のアンチセンス化合物はＬＦＡ−３をコードする核酸のいかなる部分にも特異的にハイブリダイズする（標的設定される）ように設計することができる。しかしながら、上述の結果は、ヒトＬＦＡ−３をコードする核酸の幾つかの領域及びそのような領域内の配列がヒトＬＦＡ−３のアンチセンス調節に特に好ましく、したがって、それを調節するように設計されるアンチセンス化合物の好ましい標的領域であることを示す。
【０１４６】
１．ヒトＬＦＡ−３のＡＵＧ（開始）コドン領域は、本発明の好ましい態様である、ＩＳＩＳ１６３７１及び１６９１０（配列番号２）の標的領域である。
２．ヒトＬＦＡ−３のＯＲＦ（読み取り枠）はＩＳＩＳ１６３７３、１６３７４、１６９０９及び１７１５９（配列番号４及び５）の標的領域であり、これらは全て本発明の好ましい態様である。
３．ヒトＬＦＡ−３の終止コドン領域はＩＳＩＳ１６３７６及び１６３７７（配列番号７及び８）の標的領域であり、これらは全て本発明の好ましい態様である。図１に示されるように、これらのアンチセンス化合物はヒトＬＦＡ−３配列の塩基対７６０〜８００（配列番号１）を含み、したがって、この領域（配列番号１９）はヒトＬＦＡ−３を調節するように設計されるアンチセンス化合物の好ましい標的領域である。
４．ヒトＬＦＡ−３の３’ −ＵＴＲ（非翻訳領域）は、本発明の好ましい態様であるＩＳＩＳ１６３７８（配列番号９）の標的領域である。
【０１４７】
Ｅ．ＬＦＡ−３の異なるイソ型への標的指向性：配列番号１（Ｗａｌｌｎｅｒ ｅｔ ａｌ．， Ｊ． Ｅｘｐ． Ｍｅｄ．， １９８７， １６６， ９２３）によってコードされるＬＦＡ−３の膜貫通形態に加えて、ＬＦＡ−３の２つのイソ型が公知である。本発明のアンチセンス化合物のあるものは、特定の効果を達成するために特定のイソ型を指向させることができる。
“ＰＩ連結ＬＦＡ−３” （ａ．ｋ．ａ． ＰＩ連結ＣＤ５８）は、ホスファチジルイノシトール（ＰＩ）への共有結合によって細胞膜に固定されるＬＦＡ−３のイソ型である。ＰＩ連結ＬＦＡ−３は細胞表面上に提示され、したがって細胞：細胞会合に寄与し得るが、細胞膜を横切ることがなく、したがって、おそらくはシグナル伝達を行うことはない。ＰＩ連結ＬＦＡ−３をコードするｃＤＮＡのヌクレオチド配列（Ｓｅｅｄ， Ｎａｔｕｒｅ， １９８７， ３２９， ８４０）は、７１９〜１０４０位で膜貫通イソ型をコードするもの（配列番号１）から分かれる。したがって、ＩＳＩＳ１６３７６、１６３７７及び１６３７８（配列番号７、８及び９）を含むがこれらに限定されるものではない、この領域に向けて標的設定されるアンチセンス化合物は、ＰＩ連結ＬＦＡ−３の発現に大きな衝撃を与えることなく膜貫通形態の発現を減少又は阻害することが予想される。
【０１４８】
“可溶性ＬＦＡ−３” （ａ．ｋ．ａ． ｓＬＦＡ−３又はｓＣＤ５８）はヒトの血清及び尿並びに幾つかのヒト細胞系の培養上清において同定されている（Ｈｏｆｆｍａｎ ｅｔ ａｌ．， Ｅｕｒ． Ｊ． Ｉｍｍｕｎｏｌ．， １９９３， ２３， ３００３）。ｓＬＦＡ−３が産生されるイン・ビボ機構は未だ特徴付けられていないが、同様の生物学的な系に基づくと、（１）膜貫通ドメインを欠くイソ型をコードするｍＲＮＡを産生する、ＬＦＡ−３をコードするＲＮＡの代替スプライシング又は （２）膜結合ＬＦＡ−３タンパク質のタンパク分解性開裂のいずれかから生じる可能性がある。いかなる特定の理論によっても結び付けることを望むものではないが、ｓＬＦＡ−３が代替スプライシングによって産生される場合、ＬＦＡ−３の膜貫通ドメインをコードするＲＮＡの部分に向けて標的設定されているアンチセンス化合物は、ｓＬＦＡ−３の発現に大きな衝撃を与えることなくＬＦＡ−３の膜貫通形態の発現を減少又は阻害することが予想される。膜貫通ドメインは配列番号１の塩基６５５〜１０４０によってコードされ（Ｗａｌｌｎｅｒ ｅｔ ａｌ．， Ｊ． Ｅｘｐ． Ｍｅｄ．， １９８７， １６６， ９２３）、この領域に向けて特異的に標的設定されるアンチセンス化合物にはＩＳＩＳ１６３７６、１６３７７及び１６３７８（配列番号７、８及び９）が含まれるが、これらに限定されるものではない。このようなアンチセンス化合物は、可溶性ＬＦＡ−３の発現に大きな衝撃を与えることなく、代替ＲＮＡスプライシングによって産生されるＬＦＡ−３の膜貫通形態の発現を減少又は阻害することが予想される。
【０１４９】
実施例５：Ｔ細胞刺激のアンチセンス介在阻害
研究から、ヒト内皮細胞（ＥＣ）が、フィトヘマグルチニン（ＰＨＡ）によって活性化されたＴ細胞からのインターロイキン２（ＩＬ−２）、インターロイキン４（ＩＬ−４）及びインターフェロン−ガンマ（ＩＦＮ−γ）の産生を有効に同時刺激することが示されている。ＬＦＡ−３に対するモノクローナル抗体を用いる遮断実験により、ＬＦＡ−３：ＣＤ−２介在シグナル伝達がＥＣによる同時刺激の約５０％に寄与することが示唆されている（Ｈｕｇｈｅｓ ｅｔ ａｌ．， Ｊ． Ｅｘｐ． Ｍｅｄ．， １９９０， １７１， １４５３）。サイトカインの産生及びＴ細胞の増殖によって測定される、Ｔ細胞を活性化する細胞の能力を調節する本発明のアンチセンス化合物の能力を評価するため、以下の実験を行った。
【０１５０】
Ａ．ＰＨＡ同時刺激検定：連続的に培養したＨＵＶＥＣ細胞を、６ウェル組織ＦＡＬＣＯＮ（登録商標）培養プレート（Ｂｅｃｔｏｎ−Ｄｉｃｋｉｎｓｏｎ ａｎｄ Ｃｏ．、Ｆｒａｎｋｌｉｎ Ｌａｋｅｓ、ＮＪ）において、ＬＩＰＯＦＥＣＴＩＮ（登録商標）を用いて、前に実施例に記載されるように、２５ｎＭのＩＳＩＳ１６３７４（活性ＬＦＡ−３オリゴヌクレオチド）又は１７０９２（１６３７４のスクランブル化対照）で処理した。７０時間後、処理細胞の試料をＬＦＡ−３の表面発現について、ＦＡＣＳ（登録商標）分析により、前の実施例のように、ＦＩＴＣ結合抗ＣＤ５８（Ｉｍｍｕｎｏｔｅｃｈ、Ｗｅｓｔｂｒｏｏｋ 、ＭＥ）を用いて分析した。ＩＳＩＳ１６３７４処理ＨＵＶＥＣ細胞上でのＬＦＡ−３の表面発現は、スクランブル化対照オリゴヌクレオチドＩＳＩＳ１７０９２で処理した細胞の表面上に発現するものの２０〜２５％に減少した。残りの細胞は、以下の同時刺激検定のため、組織培養丸底９６ウェルプレートに再塗布した。
【０１５１】
ＣＤ４^＋ Ｔ細胞を成人ボランティア・ドナーに由来する末梢血単球（ＰＢＭＣ）からネガティブ選択により単離したところ、得られた細胞は約９０％のＣＤ４^＋ であった。約２，０００の精製ＣＤ４^＋ Ｔ細胞を、各ウェルの、１０％ウシ胎児血清（ＦＣＳ）、２．５ｍＭグルタミン、１００Ｕ／ｍｌペニシリン及び１００μｇ／ｍＬストレプトマイシン（全て Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓから）を最終容積２００μＬで含むＲＰＭＩ １６４０培地（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ， Ｉｎｃ． 、Ｇａｉｔｈｅｒｓｂｕｒｇ、ＭＤ）に添加した。Ｔ細胞分裂促進因子フィトヘマグルチニン（Ｓｉｇｍａ Ｃｈｅｍｉｃａｌ Ｃｏ．、Ｓｔ． Ｌｏｕｉｓ 、ＭＯからのＰＨＡ−Ｌ）も３μｇ／ｍＬで培地に添加した。
【０１５２】
比較の目的で、遮断剤ＩＥ６（抗ＣＤ５８、アイソタイプＩｇＧ１、Ｂｉｏｇｅｎ、Ｃａｍｂｒｉｄｇｅ 、ＭＡからの贈り物）を５０μｇ／ｍＬで添加した；対照として、Ｋ１６／１６（非結合性ＩｇＧ１、腹水）を１：１０００希釈で添加した。他の抗ＣＤ５８遮断抗体、例えば、Ｐｅｒｃｅｐｔｉｖｅ Ｄｉａｇｎｏｓｔｉｃｓ、Ｃａｍｂｒｉｄｇｅ 、ＭＡからのカタログ番号８−４７５８Ｎ、又は Ｅｕｒｏｐｅａｎ Ｃｏｌｌｅｃｔｉｏｎ ｏｆ Ａｎｉｍａｌ Ｃｅｌｌ Ｃｕｌｔｕｒｅｓ、Ｓａｌｉｓｂｕｒｙ 、Ｕ．Ｋ．の寄託番号９１０６０５５８を用いることができる。２４時間後に培地を集め、ヒトＩＬ−２、ＩＦＮ−γ及びＩＬ−４ ＥＬＩＳＡキット（Ｃｏｕｌｔｅｒ Ｃｏｒｐ． 、Ｍｉａｍｉ 、ＦＬ）を用いて様々なＴ細胞産生サイトカインについて検定した。
【０１５３】
これらの結果が図２に示されており、ここで各データ点は２つのウェルの平均を表す。スクランブル化対照（ＳＣ）オリゴヌクレオチドＩＳＩＳ１７０９２は３種類のサイトカインの産生に対して効果がなく（“ＳＣ／ＥＣ” 、白抜きの四角）、かつサイトカインの産生を減少させる、ＬＦＡ−３もしくはＣＤ２に対するモノクローナル抗体の能力を阻害することがない（“ＳＣ／ＥＣ” 、それぞれ斜線付き及び塗りつぶした四角）。対照的に、活性アンチセンスオリゴヌクレオチド（ＡＳＯ）ＩＳＩＳ１６３７４は、サイトカイン産生の阻害において、ＬＦＡ−３もしくはＣＤ２に対する抗体とほぼ同様に有効であった（“ＡＳＯ／ＥＣ” 、白塗りの四角）。ＩＳＩＳ１６３７４を抗体と組み合わせたとき、サイトカイン産生のさらなる減少は見られなかった（“ＡＳＯ／ＥＣ” 、斜線付き及び塗りつぶした四角）。
【０１５４】
Ｂ．アロ反応：従来の研究により、ＩＦＮ−γ処理培養ＥＣが同種ＣＤ４^＋ 細胞を活性化してサイトカインを分泌させ、かつ増殖させることが可能であり、及びＬＦＡ−３に対するモノクローナル抗体がこのアロ反応を阻害することが示されている（Ｓａｖａｇｅ ｅｔ ａｌ．， Ｔｒａｎｓｐｌａｎｔａｔｉｏｎ， １９９３， ５６， １２８ ）。同時刺激を平底プレートで行い、かつＰＨＡを添加しなかったことを除いて、ＰＨＡ同時刺激検定と同様の方法でアロ反応を開始した。ＣＤ４^＋ Ｔ細胞の添加の４日前［ｄ（−４）］にＥＣを低集密で塗布し、直ちに５００Ｕ／ｍＬヒトＩＦＮ−γで処理してクラスＩＩ ＭＨＣの発現を誘導した。ｄ（−３）にＥＣをオリゴヌクレオチドで処理し、ｄ（−１）に再塗布し、ｄ０に１０μｇ／ｍＬのミトマイシンＣ（Ｓｉｇｍａ ）で１時間処理して洗浄した後、３×１０^５ 個の精製ＣＤ４^＋ Ｔ細胞を添加した。
【０１５５】
ＥＬＩＳＡキットを用いるサイトカイン検定のため、ｄ１、ｄ２又はｄ３にこれらの同時培養物から培地を集めた。ＩＬ−２を測定する実験においては、培地からの細胞のＩＬ−２の取り込みを防止するため、抗ＴＡＣを１０ｍｇ／ｍＬで添加した。抗ＴＡＣは、ＩＬ−２受容体のアルファ鎖に結合してそれがＩＬ−２と相互作用するのを遮断するモノクローナル抗体である（Ｔｏｍ Ｗａｌｄｍａｎｎからの贈り物であり、ＢｉｏＳｏｕｒｃｅ Ｉｎｔｅｒｎａｔｉｏｎａｌ 、Ｃａｍａｒｉｌｌｏ 、ＣＡから商業的に入手可能である）。加えて、ｄ４、ｄ５、ｄ６又はｄ７の同時培養の最後の１８〜２４時間の間に、１μＣｉの［^３ Ｈ］チミジンを各ウェルに添加し、Ｔ細胞の増殖を［^３ Ｈ］の取り込みによって評価した。これらのプレートを９６ウェル収穫器（Ｔｏｍｔｅｃ Ｃｏ． Ｌｔｄ． 、東京、日本）で収穫し、Ｍｉｃｒｏｂｅｔａ シンチレーション・カウンター（ＥＣ＆Ｇ Ｗａｌｌａｃ 、Ｔｕｒｋｕ 、フィンランド）でカウントした。これらの結果が図３及び４に示されており、ここで各データ点は３つの複製ウェルの平均を表す。
【０１５６】
図３に示されるように、活性アンチセンス・オリゴヌクレオチドＩＳＩＳ１６３７４でＥＣを前処理する（閉じた四角）ことで、スクランブルした対照オリゴヌクレオチド１７０９２（開いた四角）と比較してＩＬ−２及びＩＦＮ−γの産生が阻害された。ＩＦＮ−γに対する効果は著しいものではなかったが、ＩＬ−２に対するＩＳＩＳ１６３７４は約５０％阻害のレベルを少なくとも３日間維持した。
【０１５７】
３つの独立した増殖検定からの結果が図４に示されている。アロ応答は幾らか変動する；ピーク増殖応答の阻害の程度は約２０％〜約５０％の範囲であった。しかしながら、全ての場合において、ピーク増殖応答はＩＳＩＳ１６３７４でのＥＣの前処理によって阻害される。
これらの結果は、本発明のアンチセンス化合物がヒト内皮細胞上でのＬＦＡ−３の発現を特異的に阻害し、かつそのような処理がＴ細胞を活性化するそのような細胞の能力を少なくともＬＦＡ−３もしくはＣＤ２に対するモノクローナル抗体と同じ程度まで低下させることを示す。
【０１５８】
実施例６：治療方法
本発明のアンチセンス化合物はイン・ビボで免疫抑制性及び抗炎症性効果を生じ、当業者がそのような効果を必要とする、ヒトを含む動物に予防的、一時緩和的及び治療上の利益をもたらすのに用いることができる。また、本発明のアンチセンス化合物は癌細胞の転移を阻害するそれらの能力についても評価され、過剰増殖性疾患からの予防的、一時緩和的又は治療的軽減をもたらすのに用いられる。本発明のアンチセンス化合物を用いる治療方法には以下の例が含まれるが、これらに限定されるものではない。
【０１５９】
Ａ．望ましくない免疫応答性現象の調節：本発明は、動物において直接もしくは間接的にＬＦＡ−３が介在し、又はそれによって影響を受ける免疫応答性現象を調節する方法を提供する。このような免疫応答性現象は望ましくない効果、例えば、炎症につながり得る。ＬＦＡ−３が介在し、又はそれによって影響を受ける免疫応答性現象の調節方法は、必要であれば医薬製剤中の、１種類以上の本発明のアンチセンス化合物（又はそれらと１種類以上の抗炎症剤もしくは免疫抑制性非アンチセンスベースの薬剤、すなわちＮＡＢＡの組み合わせ；下記参照）を動物に投与することを含む。本発明のアンチセンス化合物の幾つかの特定の治療様式が例として以下に続くが、これらは本発明の範囲を限定しようとするものではない。
【０１６０】
１．ＬＦＡ−３及びＣＤ２が介在する現象：本発明はＬＦＡ−３の発現を阻害するための組成物及び方法を提供する。引き続く分子及び／又は細胞の現象を誘発するにはＬＦＡ−３のＣＤ２への結合が必要であるため、ＬＦＡ−３発現の阻害はそのような現象の減少又は阻害を生じるものと予想される。様々なイン・ビトロ及びイン・ビボ法が、本発明のアンチセンス化合物のＬＦＡ−３：ＣＤ２相互作用を阻害する能力の検定に利用可能である （例えば、Ｄｕｓｔｉｎ ｅｔ ａｌ．， Ｊ． Ｃｅｌｌ Ｂｉｏｌ．， １９９６， １３２， ４６５；Ｄｉｎｇ ｅｔ ａｌ．， Ｊ． Ｉｍｍｕｎｏｌ．， １９９６， １５７， １８６３ を参照）。
【０１６１】
２．Ｔ細胞の活性化：本発明は、動物においてＬＦＡ−３介在Ｔ細胞活性化を調節する方法であって、必要であれば医薬製剤中の、１種類以上の本発明のアンチセンス化合物、又はそれらと１種類以上の抗炎症剤もしくは免疫抑制剤との組み合わせを動物に投与することを含む方法を提供する。膜結合ＣＤ２について細胞表面ＬＦＡ−３と競合する可溶性形態のＬＦＡ−３は、Ｔ細胞の活性化を妨害する（Ｙａｍａｓｈｉｔａ ｅｔ ａｌ．， Ｉｍｍｕｎｏｌ．， １９９７， ９２， ３９）。望ましくないＬＦＡ−３介在又は関連Ｔ細胞活性化は、多くの傷害性免疫応答における重要な成分であるものと考えられ、この傷害性免疫応答には炎症、喘息、アルコール性肝硬変を含む慢性肝疾患等が含まれる（Ｍｅｎｇｅｌｅｒｓ ｅｔ ａｌ．， Ａｍ． Ｊ． Ｒｅｓｐｉｒ． Ｃｒｉｔ． Ｃａｒｅ Ｍｅｄ．， １９９４， １４９， ３４５；Ｈｏｆｆｍａｎ ｅｔ ａｌ．， Ｊ． Ｈｅｐａｔｏｌ．， １９９６， ２５， ４６５；Ｓａｎｔｏｓ−Ｐｅｒｅｚ ｅｔ ａｌ．， Ｉｍｍｕｎｏｌ． Ｌｅｔｔ．， １９９６， ５０， １７９）。必要であれば適切な医薬組成物の一部として、本発明のアンチセンス化合物を動物に投与することで、Ｔ細胞の活性化及びそれに続く望ましくない免疫応答性現象、例えば、炎症及び炎症性損傷の阻害が期待される。このような治療は、１種類以上の抗炎症剤及び／又は免疫抑制性ＮＡＢＡ及び、それに加えて、又はその代わりに、例えばＣＡＭタンパク質、Ｂ７ タンパク質、もしくはプロテインキナーゼＣに向けて標的設定された１種類以上のさらなるアンチセンス化合物との組み合わせであり得る（下記参照）。このような投与は全身性のものであっても、又は直接炎症及び／又は炎症性損傷の部位（１ヶ所もしくは複数箇所）に対するものであってもよい。本発明のアンチセンス化合物は、Ｔ細胞の活性化及び引き続く望ましくない炎症及び／又は炎症性損傷を調節するそれらの能力について、例えば、実施例５に記載される検定及び／又は他の適切なものを用いて、イン・ビトロ又はイン・ビボモデルにおいて評価される。
【０１６２】
３．同種移植片拒絶及びＧＶＨＤ：本発明は、動物における移植片対宿主病（ＧＶＨＤ）の治療又は防止を含む同種移植片拒絶を回避する方法であって、必要であれば医薬製剤中の、１種類以上の本発明のアンチセンス化合物、又はそれらと１種類以上の抗炎症剤もしくは免疫抑制剤との組み合わせを動物に投与することを含む方法も提供する。可溶性ＬＦＡ−３−ＩｇＧ１融合タンパク質によるＬＦＡ−３：ＣＤ２相互作用の阻害は、霊長類の心臓同種移植の生存期間を長期化し、免疫不全マウス／ヒトキメラにおいて異種移植された皮膚組織を保護することが示されている（Ｋａｐｌｏｎ ｅｔ ａｌ．， Ｔｒａｎｓｐｌａｎｔａｔｉｏｎ， １９９６， ６１， ３５６ ；Ｓｕｌｔａｎ ｅｔ ａｌ．， Ｎａｔｕｒｅ Ｂｉｏｔｅｃｈ．， １９９７， １５， ７５９ ）。したがって、１種類以上の本発明のＬＦＡ−３調節性アンチセンス化合物を（必要であれば、他の薬剤と組み合わせて、及び適切な医薬組成物の一部として）動物に投与することで、異種移植片もしくは同種移植片拒絶及びＧＶＨＤの調節が期待される。
【０１６３】
この方法における本発明のアンチセンス化合物の投与は全身性のものであっても、直接移植した組織（１つもしくは複数）もしくは臓器（１つもしくは複数）の領域（１ヶ所もしくは複数箇所）に対するものであってもよく、又はエキソ・ビボで移植以前に組織（１つもしくは複数）もしくは臓器（１つもしくは複数）に投与される。このような治療は、１種類以上の抗炎症剤及び／又は免疫抑制性ＮＡＢＡ及び、それに加えて、又はその代わりに、ＣＡＭタンパク質、Ｂ７ タンパク質、もしくはプロテインキナーゼＣに向けて標的設定された１種類以上のさらなるアンチセンス化合物との組み合わせであり得る（下記参照）。本発明のアンチセンス化合物は、同種移植片拒絶を調節するそれらの能力について、１種類以上の当該技術分野において公知の検定及び／又は１種類以上の適切な動物モデルを用いて評価される（例えば、Ｓｔｅｐｋｏｗｓｋｉ ｅｔ ａｌ．， Ｊ． Ｉｍｍｕｎｏｌ．， １９９４， １５３， ５３３６ 、及び Ｂｅｎｎｅｔｔらの米国特許第 ５，５１４，７８８号の実施例２１を参照、これらは参照することによりここに組み込まれる）。
【０１６４】
５．関節炎：本発明は、動物における様々な形態の関節炎を治療する方法であって、必要であれば医薬製剤中の、１種類以上の本発明のアンチセンス化合物、又はそれらと１種類以上の抗炎症剤もしくは免疫抑制剤との組み合わせを動物に投与することを含む方法も提供する。このような投与は全身性のものであっても、直接関与する組織、例えば、滑液に対するものであってもよい。可溶性形態のＬＦＡ−３（ＣＤ５８）は関節リューマチ（ＲＡ）の患者の滑液中で同定されており、かつＲＡではないヒトにおいて見出される濃度と比較してＲＡ患者においては低い濃度で見出される（Ｈｏｆｆｍａｎ ｅｔ ａｌ．， Ｃｌｉｎ． Ｅｘｐ． Ｉｍｍｕｎｏｌ．， １９９６， １０４， ４６０ ；Ｈｏｆｆｍａｎ ｅｔ ａｌ．， Ｃｌｉｎ． Ｅｘｐ． Ｒｈｅｕｍａｔｏｌ．， １９９６， １４， ２３ ）。したがって、１種類以上の本発明のＬＦＡ−３調節性アンチセンス化合物を（必要であれば、他の薬剤と組み合わせて、及び適切な医薬組成物の一部として）動物に投与することで、ＲＡの調節が期待される。
【０１６５】
ＲＡを治療するための本発明のアンチセンス化合物の投与は、１種類以上のさらなるアンチセンス化合物又は抗炎症剤／免疫抑制性ＮＡＢＡとの組み合わせであってもよい（下記参照）。本発明のアンチセンス化合物は、関節炎及びそれらから生じる炎症性損傷を調節するそれらの能力について、当該技術分野において公知の１種類以上の検定及び／又は１種類以上の適切な動物モデルを用いて評価される（例えば、Ｂｅｎｏｉｓｔ らの公開ＰＣＴ出願 ＷＯ ９５／３２２８５を参照）。
【０１６６】
６．腸の炎症性障害：本発明は、動物における様々な腸の炎症性障害を治療する方法であって、必要であれば医薬製剤中の、１種類本発明のアンチセンス化合物、又はそれらと１種類以上の抗炎症剤もしくは免疫抑制剤との組み合わせを動物に投与することを含む方法も提供する。このような障害には、例えば、クローン病（ＣＤ）及び他の形態の局所的腸炎；並びに潰瘍性大腸炎（ＵＣ）及び肉芽腫性、虚血性及び放射線大腸炎を含む様々な形態の大腸炎が含まれる（Ｔｈｅ Ｍｅｒｃｋ Ｍａｎｕａｌ ｏｆ Ｄｉａｇｎｏｓｉｓ ａｎｄ Ｔｈｅｒａｐｙ， １５ｔｈ Ｅｄ．， ｐｐ． ７９７−８０６， Ｂｅｒｋｏｗ ｅｔ ａｌ．， ｅｄｓ．， Ｒａｈａｙ， Ｎ．Ｊ．， １９８７を参照）。クローン病に見出される組織肉芽腫に類似する細胞性の凝集体が形成されるクローン病のイン・ビトロモデルが記述されている。ＬＦＡ−３に対する抗体を遮断することで、このモデルにおけるイン・ビトロ凝集体形成が阻害される（Ｍｉｓｈｒａ ｅｔ ａｌ．， Ｇａｓｔｒｏｅｎｔｅｒｏｌ．， １９９３， １０４， ７７２ ）。さらに、ＲＡの場合と同様に、炎症性腸疾患の患者においては可溶性ＬＦＡ−３の濃度が低下する（Ｈｏｆｆｍａｎ ｅｔ ａｌ．， Ｚ． Ｇａｓｔｒｏｅｎｔｅｒｏｌ．， １９９６， ３４， ５２２）。これらの知見は、１種類以上の本発明のＬＦＡ−３調節性アンチセンス化合物を（必要であれば、他の薬剤と組み合わせて、及び適切な医薬組成物の一部として）動物に投与することで、クローン病及び他の腸の炎症性障害の調節が期待できることを示す。
【０１６７】
このような障害を治療するための本発明のアンチセンス化合物の投与は、１種類以上のさらなるアンチセンス化合物又は抗炎症剤及び／又は免疫抑制性ＮＡＢＡとの組み合わせであってもよい（下記参照）。本発明のアンチセンス化合物は、腸の炎症性障害を調節するそれらの能力について、１種類以上の当該技術分野において公知の検定及び／又は１種類以上の適切な動物モデルを用いて評価される（例えば、Ｏｋａｙａｓｕ ｅｔ ａｌ．， Ｇａｓｔｒｏｅｎｔｅｒｏｌ．， １９９０， ９８， ６９４ ；Ｍｉｓｈｒａ ｅｔ ａｌ．， Ｇａｓｔｒｏｅｎｔｅｒｏｌ．， １９９３， １０４， ７７２ ；及び Ｂｅｎｎｅｔｔらの米国特許第 ５，５１４，７８８号の実施例２０を参照、これらは参照することによりここに組み込まれる）。
【０１６８】
７．自己免疫疾患及び障害：本発明は、自己免疫甲状腺障害；関節炎の自己免疫形態；多発性硬化症（ＭＳ）；若年性糖尿病の幾つかの形態；重症筋無力症；尋常性天疱瘡；及び全身性エリテマトーデス（ＳＬＥもしくはループス）を含むがこれらに限定されるものではない様々な自己免疫疾患及び障害の治療方法も提供する（自己免疫障害の再検討のためには、Ｓｔｅｉｎｍａｎ， Ｓｃｉ． Ａｍｅｒ．， １９９３， ２６９， １０７を参照）。本発明の好ましい態様は、自己免疫甲状腺障害、例えば、グレーブス病（甲状腺中毒症）、橋本病及びド・ケルヴァン甲状腺炎の治療又は防止に関与し、これはＬＦＡ−３がこのような状態において発現することが観察されているためである（Ｚｈｅｎｇ ｅｔ ａｌ．， Ｊ． Ａｕｔｏｉｍｍｕｎ．， １９９０， ３， ７２７ ）。必要であれば適切な医薬組成物の一部として、本発明のアンチセンス化合物を動物に投与することで、自己免疫疾患の発症及び引き続く望ましくない現象の防止又は阻害が期待される。このような治療は、１種類以上の抗炎症剤／免疫抑制性ＮＡＢＡ並びに、それに加えて、又はその代わりに、例えばＣＡＭタンパク質、Ｂ７ タンパク質、もしくはプロテインキナーゼＣに向けて標的設定されている、１種類以上のさらなるアンチセンス化合物との組み合わせであってもよい。このような投与は、障害の性質に応じて、全身性のものであっても直接特定の組織に対するものであってもよい。例えば、全身性投与はＳＬＥに対してより適切であり、これに対してグレーブス病の場合には甲状腺又は隣接組織への直接投与がより有効であろう。本発明のアンチセンス化合物は、自己免疫疾患を防止又は阻害するそれらの能力について、当業者に公知の適切な検定及び動物モデルを用いて評価される（例えば、Ｂｕｒｋｈａｒｄｔ ｅｔ ａｌ．， Ｒｈｅｕｍａｔｏｌ． Ｉｎｔ．， １９９７， １７， ９１ を参照）。
【０１６９】
Ｂ．過剰増殖性障害の治療：良性腫瘍、及び発達過程の早期に検出されている原発性悪性腫瘍を有する患者は、しばしば、その良性もしくは原発性腫瘍を外科的に除去することによってうまく治療することができる。しかしながら、検査をしないと、悪性腫瘍に由来する細胞が浸潤及び転移のプロセスにより患者の身体中に広がる。浸潤は、最初の付着部位から脱着し、例えば、下層をなす基底膜に侵入する癌細胞の能力を指す。転移は、（１）癌細胞がその細胞外マトリックスから脱着し、（２）脱着した癌細胞が、しばしば循環系を介して、患者の身体の別の部分に移動し、かつ（３）遠位の不適切な細胞外マトリックスに付着し、それにより二次腫瘍が生じ得る病巣を作製する、という現象の系列を示す。正常細胞は浸潤もしくは転移する能力を持たず、及び／又はそのような現象が生じたとしてもアポトーシス（プログラムされた細胞死）を受ける（Ｒｕｏｓｌａｈｔｉ， Ｓｃｉ． Ａｍｅｒ．， １９９６， ２７５， ７２）。
【０１７０】
播種性の前癌性又は癌性細胞は、しばしば、上記転移プロセスの工程（３）を促進し得る接着分子の異所的発現を示す。このような接着分子の例にはＩＣＡＭ−１及び他のＣＡＭが含まれる（再検討のためには、Ｔａｎｇ ｅｔ ａｌ．， Ｉｎｖａｓｉｏｎ Ｍｅｔａｓｔａｓｉｓ， １９９４， １４， １０９ を参照）。ＬＦＡ−３も幾つかの過剰増殖性疾患、例えば、様々な骨髄腫（Ｃｏｏｋ ｅｔ ａｌ．， Ａｃｔａ Ｈａｅｍａｔｏｌ．， １９９７， ９７， ８１ ；Ｔａｔｓｕｍｉ ｅｔ ａｌ．， Ｊｐｎ． Ｊ． Ｃａｎｃｅｒ Ｒｅｓ．， １９９６， ８７， ８３７）、膀胱腫瘍（Ｎｏｕｒｉ ｅｔ ａｌ．， １９９６， Ｕｒｏｌ． Ｉｎｔ． ５６， ６）及び成人Ｔ細胞白血球（Ｉｍａｉ ｅｔ ａｌ．， Ｉｎｔ． Ｊ． Ｃａｎｃｅｒ， １９９３， ５５， ８１１）に関連する。したがって、本発明のアンチセンス化合物を用いて１種類以上のＣＡＭを調節することで、遠位の及び／又は不適切なマトリックスに付着する播種性癌細胞の能力を低下させ、それにより原発性腫瘍の転移を調節することができる。
【０１７１】
したがって、本発明は、動物における転移を調節又は防止する方法であって、必要であれば医薬製剤中の、１種類以上の本発明のアンチセンス化合物を動物に投与することを含む方法も提供する。このような治療は、１種類以上のさらなるアンチセンス化合物又は抗癌化学療法用ＮＡＢＡとの組み合わせであってもよい（下記参照）。本発明のアンチセンス化合物は、転移を調節するそれらの能力について、１種類以上の当該技術分野において公知の検定及び／又は１種類以上の適切な動物モデルを用いて評価される（例えば、Ｂｅｎｎｅｔｔ らの米国特許第 ５，５１４，７８８号の実施例１６〜１８を参照、これは参照することによりここに組み込まれる）。
【０１７２】
Ｃ．病原体によって引き起こされる疾患の治療：幾つかの研究により、ＬＦＡ−３の発現及び／又は機能がヒトウイルスの病原性の強化と関連付けられている。例えば、ＬＦＡ−３はサイトメガロウイルスに感染した線維芽細胞において上方調節され（Ｇｒｕｎｄｙ ｅｔ ａｌ．， Ｉｍｍｕｎｏｌ．， １９９３， ７８， ４０５）、この効果は抗ウイルス性ＮＡＢＡガンシクロビア及びフォスカーネットでの治療によっては遮断されない（Ｃｒａｉｇｅｎ ｅｔ ａｌ．， Ｔｒａｎｓｐｌａｎｔａｔｉｏｎ， １９９６， ６２， １１０２ ）。したがって、移植レシピエントにおける従来の抗ウイルス治療にもかかわらず、ＣＭＶ感染ドナー細胞がＬＦＡ−３を過剰発現し、そのためそれが事実上炎症誘発性であることがあり、その特徴は免疫病理又はＧＶＨＤもしくは同種移植片拒絶の悪化に寄与する可能性がある。イン・ビボでレシピエント動物を、及び／又はエキソ・ビボでドナー細胞もしくは組織を、必要であれば抗ウイルス性ＮＡＢＡと組み合わせて、本発明のアンチセンス化合物で治療することで、そのような望ましくない効果の防止又は制限が期待される。
【０１７３】
ヒト免疫不全ウイルス（ＨＩＶ）の場合、ＬＦＡ−３に対する可溶性抗体によるＬＦＡ−３（ＣＤ５８）の固定がＨＩＶの複製を強化することが研究によって示されている（Ｓｈａｔｔｏｃｋ ｅｔ ａｌ．， Ｊ． Ｉｎｆｅｃｔ． Ｄｉｓ．， １９９６， １７４， ５４ ）。この効果は、Ｔ細胞においてＬＦＡ−３リガンド、ＣＤ２に対する抗体がＨＩＶの末端反復配列からの転写を活性化することから、シグナル伝達機構によるものである可能性がある（Ｂｒｅｓｓｌｅｒ ｅｔ ａｌ．， Ｊ． Ｉｍｍｕｎｏｌ．， １９９１， １４７， ２２９０ ）。したがって、本発明のアンチセンス化合物で個体を治療することでＨＩＶの成長及び伝播の制限が期待される。
ＬＦＡ−３の発現は口腔扁平苔癬（ＯＬＰ）を有する患者の頬粘膜に存在する病変において増加し、したがって、ＯＬＰの病因に関連付けられている（Ｋｉｒｂｙ ｅｔ ａｌ．， Ｏｒａｌ Ｄｉｓ．， １９９５， １， １９３ ）。したがって、本発明のアンチセンス化合物で個体を治療することでＯＬＰの成長及び伝播の制限が期待される。
【０１７４】
Ｄ．組み合わせ治療及び組成物
１．アンチセンス化合物の組み合わせ：上述のように２種類以上のアンチセンス化合物を同時に投与することができる。また、組み合わせ治療は、まず（１）１種類以上のＣＡＭに向けて標的設定された１種類以上のアンチセンス化合物（又は、それらと１種類以上の抗炎症剤／免疫抑制剤もしくは化学療法剤との組み合わせ）を含む第１組成物を第１の期間投与し、次に（２）１種類以上のＣＡＭに向けて標的設定された１種類以上のアンチセンス化合物（又は、それらと１種類以上の抗炎症剤もしくは化学療法剤との組み合わせ）を含む第２組成物の投与に第２の期間“切り替える” ことにより行うこともできる。同時に投与しようと連続的に投与しようと、アンチセンス化合物の好ましい組み合わせには、細胞の接着を介在する分子、例えば：（１）ＬＦＡ−３及びＩＣＡＭ−１；（２）ＬＦＡ−３及びＶＣＡＭ−１；（３）ＬＦＡ−３及びＥＬＡＭ−１；並びに（４）ＬＦＡ−３及びＢ７タンパク質、例えば、Ｂ７−１もしくはＢ７−２に向けて標的設定されているものが含まれる。Ｂ７−１及びＬＦＡ−３は同時刺激的な様式でＴ細胞を活性化するように作用する（Ｐａｒｒａ ｅｔ ａｌ．， Ｊ． Ｉｍｍｕｎｏｌ．， １９９７， １５８， ６３７ ；Ｐａｒｒａ ｅｔ ａｌ．， Ｍｏｌ． Ｃｅｌｌ． Ｂｉｏｌ．， １７， １３１４）、組み合わせ（４）がＴ細胞活性化の調節に特に好ましい。ＩＣＡＭ−１、ＶＣＡＭ−１、ＥＬＡＭ−１及びＢ７タンパク質に向けて標的設定されているアンチセンス化合物は、全て Ｂｅｎｎｅｔｔらの米国特許第 ５，５１４，７８８号及び第 ５，５９１，６２３号並びに１９９６年１２月３１日出願の同時係属米国特許出願第 ０８／７７７，２６６ 号に記載されている。
【０１７５】
所望であれば、望ましくない障害又は疾患の妨害又は防止に必要とされるレベルを達成するため、ＬＦＡ−３（又は他のＣＡＭ）の発現の治療的アンチセンス調節をさらなる治療と組み合わせることができる。このような組み合わせは、例えば、（ａ）２種類以上のＣＡＭに向けて標的設定されている２種類以上のアンチセンス化合物、（ｂ）あるものがＣＡＭに、及び他のものが遺伝子標的に向けて標的設定されている２種類以上のアンチセンス化合物、又は（ｃ）炎症を患う動物を治療する場合、非アンチセンスベースの抗炎症剤もしくは免疫抑制剤と組み合わせた、ＣＡＭに向けて標的設定されているアンチセンス化合物を同時投与することにより行うことができる。過剰増殖性疾患もしくは障害を患う動物を治療しようとする場合、ＣＡＭに向けて標的設定されているアンチセンス化合物を非アンチセンスベースの化学療法剤と組み合わせることができる。本発明のアンチセンス化合物と共に用いる場合、そのような非アンチセンスベースの薬剤（ＮＡＢＡ）は単純な組み合わせで（例えば、ＮＡＢＡ及びアンチセンス化合物の投与）、連続的に（例えば、第１のＮＡＢＡ及びアンチセンス化合物を特定の期間投与した後、第２のＮＡＢＡ及びアンチセンス化合物を投与する）、又は１種類以上の他のそのような非アンチセンスベースの薬剤もしくは物理的処置との組み合わせで（例えば、ＮＡＢＡ及びアンチセンス化合物の投与、又は、例えば過剰増殖性障害の治療においては、放射線療法と組み合わせた１種類以上のＮＡＢＡ及びアンチセンス化合物の投与）用いることができる。治療計画において２種類（もしくはそれ以上）のアンチセンス化合物、又は１種類以上のアンチセンス化合物及び１種類以上のＮＡＢＡの組み合わせを同時に投与しようとする場合、好ましい組成物の１つは、両者の（もしくは全ての）成分を含有する脂質小胞、特には無菌的に安定化された脂質小胞を含むものである。本発明の脈絡において、“治療計画” という用語は治療、一時的緩和及び予防様式を包含しようとするものである。
【０１７６】
抗ウイルス用途については、本発明のアンチセンス化合物は関心のある特定のウイルスを指向するアンチセンス化合物と組み合わせることができる。例えば、ＣＭＶ感染細胞のイン・ビボ又はエキソ・ビボ治療に対しては、アンチセンス化合物を、ＣＭＶに向けて標的設定されている１種類以上のアンチセンス化合物と組み合わせることができる。ＣＭＶに向けて標的設定されている特に好ましいアンチセンス化合物、及びそれらを使用する治療方法は、米国特許第 ５，４４２，０４９号及び第 ５，５９５，９７８号（参照することによりここに組み込まれる）に記載されている。ＨＩＶに対する特に好ましいアンチセンス化合物は米国特許第 ５，１６６，１９５号及び第 ５，５９１，６２３号（これらも参照することによりここに組み込まれる）に記載されている。１種類以上の非アンチセンスベースの抗ウイルス剤、例えば、ガンシクロビル及びフォスカーネットも１種類以上の本発明のアンチセンス化合物と組み合わせることができる。本発明のアンチセンス化合物と好ましく組み合わせられる他の非アンチセンスベースの抗ウイルス剤には、米国特許第 ５，５２３，３８９号及び第 ５，６２７，１８５号（参照することによりここに組み込まれる）並びに公開ＰＣＴ特許第 ＷＯ ９６／４０１６４号に記載されるものが含まれる。
【０１７７】
２．化学療法剤との組み合わせ：過剰増殖性障害を治療する目的で、本発明のアンチセンス化合物を、さらに、又は代わりに、非アンチセンスベースの化学療法剤と組み合わせて用いることができる。本発明のアンチセンス化合物との組み合わせで用いることができるような薬剤の例には、ダウノルビシン、ダウノマイシン、ダクチノマイシン、ドキソルビシン、エピルビシン、イダルビシン、エソルビシン、ブレオマイシン、マホスファミド、イホスファミド、シトシンアラビノシド、ビス−クロロエチルニトロソ尿素（ｂｉｓ−ｃｈｌｏｒｏｅｔｈｙｌｎｉｔｒｏｓｕｒｅａ ）、ブスルファン、マイトマイシンＣ、アクチノマイシンＤ、ミトラマイシン、プレドニゾン、ヒドロキシプロゲステロン、テストステロン、タモキシフェン、ダカルバジン、プロカルバジン、ヘキサメチルメラミン、ペンタメチルメラミン、ミトキサントロン、アムサクリン、クロラムブシル、ナイトロジェンマスタード、メチルシクロヘキシルニトロソ尿素、メルファラン、シクロホスファミド、６−メルカプトプリン、６−チオグアニン、シタラビン（ＣＡ）、５−アザシチジン、ヒドロキシ尿素、デオキシコホルマイシン、５−フルオロウラシル（５−ＦＵ）、４−ヒドロキシペルオキシシクロホスホロアミド、５−フルオロデオキシシウリジン５−ＦＵｄＲ）、メトトレキセート（ＭＴＸ）、コルヒチン、ビンクリスチン、ビンブラスチン、エトポシド、トリメトレキセート、テニポシド、シスプラチン及びジエチルスチルベストロール（ＤＥＳ）が含まれるが、これらに限定されるものではない（一般には、Ｔｈｅ Ｍｅｒｃｋ Ｍａｎｕａｌ ｏｆ Ｄｉａｇｎｏｓｉｓ ａｎｄ Ｔｈｅｒａｐｙ， １５ｔｈ Ｅｄ．， ｐｐ． １２０６−１２２８， Ｂｅｒｋｏｗ ｅｔ ａｌ．， ｅｄｓ．， Ｒａｈａｙ， Ｎ．Ｊ．， １９８７を参照）。
【０１７８】
３．抗炎症剤／免疫抑制剤との組み合わせ：本発明のアンチセンス化合物と組み合わせて用いることができる非アンチセンスベースの抗炎症剤又は免疫抑制剤には、サリチル酸塩；インドメタシン、イブプロフェン、フェノプロフェン（ｆｅｎｏｐｏｆｅｎ ）、ケトプロフェン、ナプロキセン、ピロキシカム、フェニルブタゾン、オキシフェンブタゾン、スリンダク及びメクロフェナメートを含む非ステロイド性抗炎症薬（ＮＳＡＩＤ）；金化合物、例えば、オーラノフィン（ａｕｒａｎｏｆｉｎ ）；Ｄ−ペニシラミン；シクロホスファミド；メトトレキセート；アザチオプリン；コルヒチン；ヒドロキシクロロキン；コルチコトロピン；ステロイド及び副腎皮質ステロイド、例えば、ヒドロコルチゾン、デオキシヒドロコルチゾン、フルドロコルチゾン、プレドニゾロン、メチルプレドニゾロン、プレドニゾン、トリアムシノロン、デキサメタゾン、ベタメタゾン及びパラメタゾンが含まれるが、これらに限定されるものではない。一般には、Ｔｈｅ Ｍｅｒｃｋ Ｍａｎｕａｌ ｏｆ Ｄｉａｇｎｏｓｉｓ ａｎｄ Ｔｈｅｒａｐｙ， １５ｔｈ Ｅｄ．， ｐｐ． １２３９−１２６７ ａｎｄ ２４９７−２５０６， Ｂｅｒｋｏｗ ｅｔ ａｌ．， ｅｄｓ．， Ｒａｈａｙ， Ｎ．Ｊ．， １９８７を参照。
【図面の簡単な説明】
【図１】図１Ａ及び１Ｂは、ヒトＬＦＡ−３をコードするｃＤＮＡの配列（配列番号１；ＧｅｎＢａｎｋ受入番号Ｙ００６３６、ローカス名“ＨＳＬＦＡ３” ）及び実施例に記載されるアンチセンスオリゴヌクレオチドの位置及び配列を示す。このｃＤＮＡ配列は５’ から３’ に記載されており、１０塩基毎を示す縦のマークを有する；この配列における塩基の累積番号は各ラインの右にボールド体で示されている。開始（ＡＴＧ）及び終止（ＴＧＡ）コドンはボールド体表記され、かつ二重下線が付されている。アンチセンスオリゴヌクレオチドのヌクレオチド塩基配列はそれらの相補性を示すため３’ から５’ に記載され、それらのＩＳＩＳ番号は各ラインの右の括弧内に示されている。
【図２】ヒト内皮細胞をアンチセンスオリゴヌクレオチドで前処理することでＰＨＡ活性化ＣＤ４^＋ Ｔ細胞からのサイトカイン産生の同時刺激が阻害されることを示す。記号及び略語：“ＳＣ” 、スクランブルした対照オリゴヌクレオチド（ＩＳＩＳ１７０９２）；“ＡＳＯ” 、活性アンチセンスオリゴヌクレオチド（ＩＳＩＳ１６３７４）；白抜きの四角、モノクローナル抗体の添加なし；斜線付きの四角、ＬＦＡ−３に対するモノクローナル抗体を添加；塗りつぶした四角、ＣＤ２に対するモノクローナル抗体を添加。
【図３】ヒト内皮細胞をアンチセンスオリゴヌクレオチドで前処理することでアロジェネイックＣＤ４^＋ Ｔ細胞によるサイトカイン産生が阻害されることを示す。指示されるように、ＩＬ−２の利用を防止するため、上のパネルの実験においてはＩＬ−２受容体のアルファ鎖に対する抗体（すなわち、“抗ＴＡＣ” ）を添加した。記号：白抜きの四角、スクランブルした対照オリゴヌクレオチド（ＩＳＩＳ１７０９２）；塗りつぶした四角、活性アンチセンスオリゴヌクレオチド（ＩＳＩＳ１６３７４）。
【図４】ヒト内皮細胞をアンチセンスオリゴヌクレオチドで前処理することでアロジェネイックＣＤ４^＋ 細胞の増殖が阻害されることを示す。記号：白抜きの四角、スクランブルした対照オリゴヌクレオチド（ＩＳＩＳ１７０９２）；塗りつぶした四角、活性アンチセンスオリゴヌクレオチド（ＩＳＩＳ１６３７４）。
【配列表】

[0001]
FIELD OF THE INVENTION
The present invention provides compositions and methods for detecting and modulating lymphocyte function-associated antigen 3 (LFA-3) protein, including human LFA-3 (also known as CD58 antigen). In particular, the present invention relates to antisense compounds that can specifically hybridize with a nucleic acid encoding an LFA-3 protein.
[0002]
LFA-3 mediates specific cell-cell interactions. Therefore, modulating the expression of LFA-3 is expected to control such cell-cell interactions and the resulting effects, such as, for example, inflammation. Accordingly, the present invention is directed to diagnostic methods for detecting LFA-3 mediated processes, as well as prophylactic and therapeutic methods for preventing or inhibiting, respectively. Further, the invention is directed to treatment of conditions associated with abnormal expression of LFA-3 protein. The present invention also relates to reagents for treating, diagnosing, and researching disease states or disorders that correspond to modulation of LFA-3 protein expression. Inhibition of cell hyperproliferation and a corresponding prophylactic, palliative and therapeutic effect results from treatment with the antisense compounds of the present invention.
[0003]
BACKGROUND OF THE INVENTION
Cell-cell interactions are characteristic of various biological processes. For example, in activating the immune response, one of the earliest detectable phenomena in the normal inflammatory response is the adhesion of leukocytes to the vascular endothelium and subsequent migration of leukocytes from the vasculature to the site of infection or injury. is there. Adhesion of leukocytes to the vascular endothelium is an essential step in their migration outside the vasculature (for review, see Albelda et al., FASEB J., 1994, 8, 504). As is known in the art, cell-cell interactions are also important for the proliferation of both B and T lymphocytes, which result in enhanced humoral and cellular immune responses, respectively.
[0004]
In some cases, the adhesion of one cell type to another is mediated by interactions between specific proteins called "adhesion molecules" located on cell surface membranes. Interactions between adhesion molecules are similar to classic receptor-ligand interactions, except that the ligand is fixed on the cell surface instead of being soluble. One group of related biologically important molecules (by peptide sequences) that mediate cell-cell interactions is known in the art as CAMs (cellular adhesion molecules). CAMs include, for example, some intercellular adhesion molecules (ie, ICAM-1, ICAM-2 and ICAM-3), endothelial leukocyte adhesion molecule 1 (ELAM-1), vascular cell adhesion molecule 1 (VCAM-1) And platelet endothelial cell adhesion molecule 1 (PECAM-1). The CAM family itself is part of the immunoglobulin superfamily of genes (Newman et al., Science, 1990, 247, 1219).
In a cell: cell interaction, a given cellular adhesion molecule present on a first cell binds to one or more ligands present on a second cell. For example, ICA-1 binds to LFA-1, LFA-3 binds to CD2, and so on. Such an interaction can be expressed simply as:
cell. 1-LFA-3 + CD-cells. 2 →
cell. 1-LFA-3: CD2-cell. 2
[0005]
The binding of a given cellular adhesion molecule to its ligand can have many consequences, including (1) promoting the ability of two cells to remain in close contact for a specified period of time; The ability to pass molecules (eg, antigens) directly from one to the other and / or (2) for example, in a second cell, the conformation of a ligand of a second cell resulting from the interaction of the ligand with an adhesion molecule of the first cell. Includes the initiation of a cellular response (ie, signal transduction) due to a conformational change or chemical change. In the latter case, the free (ie, unassociated) soluble form of the appropriate adhesion molecule binds to the surface ligand of the second cell and thereby elicits the same or a similar cellular response It is possible to do. Soluble isoforms of such adhesion molecules can competitively bind to membrane-bound ligands, thereby reducing or inhibiting cell: cell interactions, and / or inhibit cell: cell de-adhesion. It can also be generated in vivo to generate it. Using LFA-3 and CD2 as examples, such a reaction can be schematically illustrated as follows, where "sCD2" refers to an excess of soluble form of CD2:
[0006]
cell. 1-LFA-3: CD2-cell. 2 + sCD2 →
cell. 1-LFA-3: sCD2 + CD2-cells. 2
As is known in the art, cell: cell interactions play an important role in the regulation of various immune responses and / or in the activation of effector cells, thymus-derived lymphocytes (T cells). That is, some T cells (eg, helper cells) produce and release other cells of the immune system, for example, factors that stimulate such other cells to produce molecular immune response activity. Acts to regulate. Other T cells [eg, cytotoxic, cytolytic or natural killer (NK) cells] directly generate immunoreactive activity, eg, by lysing target cells with foreign or abnormal antigens. In each case, T cell stimulation and antigen specificity in the immune response is mediated by cell: cell interactions between T cells and, for example, antigen presenting cells (APCs) (Bierer et al., FASEB J. , 1988, 2, 2584). At the molecular level, these cell: cell interactions are mediated by adhesion molecules present on T cells and APCs (Bierer et al., FASEB J., 1988, 2, 2584; Makgoba et al., Immunol. Today). , 1989, 10, 417).
[0007]
Several types of adhesion molecules are involved in mediating the interaction between T cells and APC. These include at least three lymphocyte function-related antigens (LFA-1, LFA-2 and LFA-3; Sanchez-Madrid et al., Proc. Natl. Acad. Sci. USA, 1982, 79, 7489) and ICAM (including ICAM-1, ICAM-2 and ICAM-3). The present invention addresses modulators of LFA-3 function, the ligand / receptor of which is CD2. CD2 is mainly expressed on T cells, including helper cells and NK cells, whereas LFA-3 is expressed on all human cells except thymocytes and some T cells (Bierer et al. , FASEB J., 1988, 2, 2584).
[0008]
LFA-3 protein is a glycoprotein expressed on the surface of various cell types (for a review of LFA-3 and related proteins, see Dustin et al., Annu. Rev. Immunol., 1991, 9, 27). LFA-3 plays a role in mediating thymocyte interaction with thymic epithelial cells and antigen-dependent and antigen-independent interactions of T lymphocytes with target cells and APCs (Wallner et al., J. Exp. Med., 1987, 166, 923). This interaction may also enhance T cell recognition of the major histocompatibility complex (MHC) (Selvaraj et al., Nature, 1987, 326, 400). LFA-3 is also associated with some hyperproliferative diseases such as myeloma (Cook et al., Acta Haematol., 1997, 97, 81; Tatsumi et al., Jpn. J. Cancer Res. , 1996, 87, 837). In addition, LFA-3 is up-regulated in cells infected with certain viruses, such as cytomegalovirus, or enhances their replication (Grundy et al., Immunol., 1993, 78, 405).
[0009]
Because LFA-3 is involved in cellular processes involved in the immune response, tumorigenesis and other disease states, inhibitors of LFA-3 expression may be useful for (1) autoimmune disorders such as multiple sclerosis, especially Autoimmune disorders of the thyroid gland, eg, Basedow's disease, and unwanted immune responses, eg, graft-versus-host disease (GVHD); (2) various inflammatory diseases or disorders involving inflammation or T cell-mediated elements, eg, Various forms of arthritis; allograft rejection; asthma; inflammatory diseases of the intestines, including Crohn's disease; various dermatological conditions, such as infections; and the like, and cancer, tumors, and their growth and spread (metastasis) Is expected to provide a novel therapeutic class of immunosuppressive and / or anti-inflammatory and / or anti-cancer agents having activity against a variety of hyperproliferative diseases or disorders, including but not limited to It is.
[0010]
To date, there are no known therapeutic agents that efficiently prevent LFA-3 expression. Current agents that affect cellular adhesion molecules include monoclonal antibodies and polypeptide soluble forms of ligands for adhesion molecules. Monoclonal antibodies to LFA-3 may prove useful in treating acute inflammatory responses by LFA-3 expression. However, binding of the antibody to membrane-bound LFA-3 mimics ligand (CD2) binding and may therefore stimulate signal transduction despite blocking of CD2 binding; Compounds that reduce or inhibit expression, eg, antisense compounds of the invention, should block both ligand and signal transduction. Furthermore, with long-term treatment, the host animal will produce antibodies to the monoclonal antibodies, thereby limiting their utility. In addition, monoclonal antibodies are large proteins and can be difficult to access to sites of inflammation. Polypeptide forms of cell adhesion molecules are subject to many of the same limitations as monoclonal antibodies, in addition to the expense of their production and their low binding affinity. Furthermore, LFA-3 is a transmembrane or membrane-bound protein, and polypeptides derived from LFA-3 are often insoluble in aqueous solutions, which limits their therapeutic potential (Dustin et al. , Annu. Rev. Immunol., 1991, 9, 27). Thus, there is a long-felt need for molecules that effectively inhibit LFA-3. Antisense oligonucleotides avoid many of the pitfalls of current agents used to block the effects of LFA-3. It has been found that such antisense compounds can regulate LFA-3 protein expression.
[0011]
[Related technology]
Published PCT patent applications WO 92/04463 and WO 92/16563 disclose monoclonal antibodies to LFA-3, such antibodies may be useful in the treatment and diagnosis of rheumatoid arthritis, autoimmune diseases and other diseases. It is shown that.
Published PCT patent applications WO 91/13981, WO 93/06852 and WO 96/33217 disclose LFA-3 proteins and fragments thereof, and the ability of these polypeptides to be used in the prevention of autoimmune diseases and graft rejection Is shown.
Published PCT Patent Application WO 93/06866 discloses a method for preventing and treating skin conditions using a non-antisense based inhibitor of the LFA-3: CD2 interaction.
Published EPO patent application EP 0 786 255 A1 discloses LFA-3 binding to CD2 or soluble forms and conjugates of CD2 binding to LFA-3, and these compounds are used to enhance allograft or xenograft tolerance. It shows that it can be used to improve.
[0012]
U.S. Pat. Nos. 4,956,281, 5,185,441 and 5,354,665 disclose nucleotide sequences encoding LFA-3 and methods for producing LFA-3 polypeptides.
U.S. Patent No. 5,547,853 discloses peptides corresponding to the CD2 binding domain of LFA-3, which have been shown to be useful for diagnostic and therapeutic purposes.
U.S. Patent No. 5,556,943 discloses sheep LFA-3 protein sequences and derivatives thereof, and indicates that such polypeptides are useful for treating leukemia.
[0013]
[Summary of the Invention]
According to the present invention, there is provided an antisense compound that specifically hybridizes with a nucleic acid encoding an LFA-3 protein. Certain antisense compounds of the present invention are designed to bind directly to mRNA translated from the gene encoding the LFA-3 protein, or to bind to a selected DNA portion thereof, thereby regulating their expression. Have been. In particular aspects of the invention, the LFA-3 protein, and the gene encoding it, are from a mammal, including a human. Also provided herein are pharmaceutical compositions comprising the antisense compounds of the invention, and various uses of the antisense compounds of the invention.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
An oligonucleotide can include a nucleotide sequence whose identity and number are sufficient to result in specific hybridization with a particular nucleic acid. Such oligonucleotides are generally described as "antisense." Antisense oligonucleotides are commonly used as research reagents, diagnostic aids and therapeutics. It has been discovered that genes encoding lymphocyte function-associated antigen-3 (LFA-3; also known as CD50 antigen), including human LFA-3, are particularly amenable to this approach. More specifically, the present invention is directed to antisense compounds, including oligonucleotides, that modulate LFA-3 expression.
[0015]
Provided herein are methods of modulating LFA-3 protein expression with antisense compounds, which may be therapeutic and diagnostic as a result of the association of LFA-3 expression with certain hyperproliferative and inflammatory diseases. It is believed to be useful. These methods are also useful, for example, as tools in detecting and determining the role of LFA-3 in various cellular functions and physiological processes and conditions, and in diagnosing conditions associated with such expression and activation. It is also useful.
[0016]
Inhibition of LFA-3 protein expression as a result of the association of LFA-3 with normal and abnormal cell-cell interactions may include, for example, various undesirable immune responsive phenomena and tumorigenicity and / or metastasis It is expected to lead to inhibition of sexual phenomena and therefore to modulate the undesirable consequences of such phenomena. Such modulation is desirable for the treatment of various inflammatory and hyperproliferative disorders or diseases (ie, to provide a prophylactic, palliative and / or therapeutic effect). Inhibition of such LFA-3, and other CAMs, further inhibits such diseases or disorders in animals suspected of, or known to be predisposed to, such diseases or disorders. Desirable to prevent or regulate the onset.
[0017]
The invention also includes a method of inhibiting various LFA-3-mediated inflammatory and tumorigenic and / or metastatic phenomena using the antisense compounds of the present invention. Also provided are methods of treating conditions in which abnormal or excessive LFA-3 expression and / or LFA-3-mediated inflammation occurs. These methods use the antisense compounds of the present invention and are believed to be useful therapeutically and as clinical research and diagnostic tools. The oligonucleotides of the invention can also be used for research purposes. Thus, the specific hybridization exhibited by the oligonucleotides of the present invention can be used in assays, purification, preparation of cell products, and in other methodologies that will be appreciated by those skilled in the art.
[0018]
The present invention uses an antisense compound which regulates the function of DNA or messenger RNA (mRNA) encoding the protein (LFA-3), and since the regulation is desirable, finally the expression of the protein is regulated. Hybridization of an antisense oligonucleotide with its mRNA target disrupts the normal role of mRNA and causes its regulation in cells. The function of the mRNA to be interfered with includes all life-supporting functions, such as translocation of the RNA to a protein translation site, actual translation of the protein from the RNA, splicing of the RNA to one or more mRNAs. Includes seeding, and perhaps even independent catalytic activity that may be involved by the RNA. The overall effect of such disruption of mRNA function is the modulation of protein expression, where "modulation" means either increasing (stimulating) or decreasing (inhibiting) the expression of the protein. In the context of the present invention, inhibition is a preferred form of regulation of gene expression.
[0019]
Preferably, certain genes are targeted for antisense attack. "Targeting" an oligonucleotide to a related nucleic acid is a multi-step process in the context of the present invention. This process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This can be, for example, a cellular gene (or mRNA transcribed from that gene) whose expression is associated with a particular disorder or disease state, or a foreign nucleic acid derived from an infectious agent. In the present invention, the target is a cellular gene associated with a hyperproliferative disorder. This targeting process also involves determining the site (s) within the gene to cause an oligonucleotide interaction to produce the desired effect, either detection or regulation of protein expression. It is. Once the target site (s) has been determined, oligos that are sufficiently complementary to the target, ie, specifically hybridize and have sufficient specificity, to produce the desired effect Select nucleotides. In general, there are several regions of the gene that can be targeted for antisense regulation: the 5 '"cap", which is an N7-cap that binds to the most 5' residue of the mRNA by a triphosphate linkage. Including methylated guanosine (Baker, Chapter 3 In: Antisense Research and Applications, Crooke et al., Eds., CRC Press, Boca Raton, FL, 1993, pages 37-53; non-translated; 37-53; 5′-UTR ”), translation initiation codon region (hereinafter“ AUG ”region), open reading frame (hereinafter“ ORF ”) or“ coding region ”, translation termination codon region (hereinafter“ stop codon ”region or abbreviated) Simply "termination") and the 3 'untranslated region Below, "3 '-UTR"). As is known in the art, these regions are arranged in a typical messenger mRNA molecule in the following order (left to right, 5 'to 3'): cap, 5'-UTR, AUG, ORF. , Stop codon, 3'-UTR. As is known in the art, some eukaryotic transcripts are translated directly, but many ORFs contain one or more sequences known as "introns," which are translated before it is translated. The expressed (non-excised) portion of the ORF is referred to as the "exon" (Alberts et al., Molecular Biology of the Cell, 1983, Galland Publishing Inc., New York, 11 pp. 11-4). . In addition, because many eukaryotic ORFs are over 1000 nucleotides in length, it is often convenient to subdivide the ORF into, for example, a 5 'ORF region, a central ORF region, and a 3' ORF region. In some cases, the ORF contains one or more sites that can be targeted for some functional significance in vivo. Examples of the latter type of site include intragenic stem-loop structures (see, for example, US Pat. No. 5,512,438) and, in native mRNA molecules, intron / exon splice sites.
[0020]
Within the context of the present invention, one preferred intragenic site is the region encompassing the translation initiation codon of the open reading frame (ORF) of the gene. As is known in the art, the translation initiation codon is typically 5'-AUG (in the translated mRNA molecule; 5'-ATG in the corresponding DNA molecule). Also called "AUG codon", "start codon" or "AUG start codon". A few genes have translation initiation codons with the RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA has been shown to function in vivo. Further, 5'-UUU functions as a translation initiation codon in vitro (Brigstock et al., Growth Factors, 1990, 4, 45; Gelbert et al., Somat. Cell. Mol. Genet., 1990, 16, .... 173; Gold and Stormo, in: Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol 2, 1987, Neidhardt et al, eds, American Society for Microbiology, Washington, D.C., p 1303). Thus, the terms "translation initiation codon" and "initiation codon" refer to a number of codon sequences, although the starting amino acid in each case is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). May be included. Eukaryotic and prokaryotic genes may have two or more alternative start codons, both of which may be in a particular cell type or tissue to produce related polypeptides having different amino terminal sequences, or It is also known in the art that it can be used preferentially for translation initiation under a particular set of conditions. In the context of the present invention, “initiation codon” and “translation initiation codon” are transcribed in vivo from the gene encoding the LFA-3 protein, regardless of the nucleotide sequence (s) of such codon. Refers to the codon (s) used to initiate translation of the generated mRNA molecule. The gene has three sequences of translation termination codons (ie, "stop codons"), namely, 5'-UAA, 5'-UAG, and 5'-UGA (the corresponding DNA sequences are 5'-TAA, 5'-respectively, respectively). (TAG and 5'-TGA) is also known in the art. The terms "initiation codon region" and "translation initiation region" refer to an mRNA or gene that includes about 50 contiguous (adjacent) nucleotides in either direction (ie, 5 'or 3') from the translation initiation codon. Refers to the department. Similarly, the terms "stop codon region" and "translation termination region" are used to refer to mRNA or such that it contains about 50 contiguous (contiguous) nucleotides in either direction (ie, 5 'or 3') from the translation stop codon. Refers to a part of a gene.
The remainder of this detailed description is based on (1) antisense compounds of the invention, and (2) bioequivalents, and (3) their exemplary utility, as well as (4) antisense compounds of the invention. Pharmaceutical compositions containing the compounds, and (5) their method of administration, are more fully described.
[0021]
1. Antisense compounds of the invention: The invention uses antisense compounds that modulate LFA-3 protein. The term “antisense compound” refers to (a) a nucleic acid encoding a nucleic acid encoding a LFA-3 protein, in which a synthetic oligonucleotide having a nucleobase sequence capable of specifically hybridizing is added to a peptide nucleic acid (PNA). And (b) specifically excludes ribozymes and nucleic acids of biological origin. In the context of the present invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or a mimetic thereof. The term includes oligonucleotides containing non-natural moieties that function similarly, as well as oligonucleotides that contain natural nucleobases, sugars and covalent intersugar (backbone) linkages. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties, such as enhanced cellular uptake, increased affinity for nucleic acid targets, and increased stability in the presence of nucleases. The antisense compounds of the present invention are synthesized in vitro and do not include antisense compositions of biological origin or genetic vector constructs designed to direct in vivo synthesis of antisense molecules. .
[0022]
The antisense compounds according to the present invention preferably contain about 8 to about 30 nucleobases, more preferably about 12 to about 28, most preferably about 20 to about 26 nucleobases. Particularly preferred antisense compounds are antisense oligonucleotides. A discussion of antisense oligonucleotides and some desirable modifications can be found in De Mesmaeker et al. , Acc. Chem. Res. , 1995, 28, 366.
Oligonucleotides are polymers of repeating units commonly known as nucleotides. Unmodified (natural) nucleotides have three components: (1) a nitrogen-containing heterocyclic base, which is linked to (2) a 5-pentofuranosyl sugar by one of its nitrogen atoms; 2.) A phosphoric acid esterified to one of the 5 'or 3' carbon atoms of the sugar. When incorporated into an oligonucleotide chain, the phosphate of the first nucleotide is also esterified by a 3'-5 'phosphate linkage to the adjacent sugar of the second adjacent molecule.
[0023]
As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is usually a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. Nucleotides are nucleosides that further contain a phosphate group covalently linked to its own sugar moiety. For nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2 ', 3' or 5 'hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. The respective ends of the linear polymer structure can be further joined to form a cyclic structure, but in the context of the present invention, open linear structures are generally preferred.
[0024]
Within this oligonucleotide structure, the phosphate groups are said to form the intersugar "backbone" of the oligonucleotide. The usual linkage or backbone of RNA and DNA is a 3 'to 5' phosphodiester linkage. The backbone of an oligonucleotide (or other antisense compound) places a series of bases in a particular order; usually, unless otherwise indicated, the order of this ordered base, described in the order 5 'to 3', The descriptive designation of the sequence is known as the nucleotide or nucleobase sequence.
[0025]
Oligonucleotides can include nucleotide sequences whose identity and number are sufficient to cause specific hybridization with a particular nucleic acid. Oligonucleotides that specifically hybridize to a portion of the sense strand of the gene are generally described as "antisense." In the context of the present invention, "hybridization" refers to hydrogen bonding between complementary nucleotides, which may be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that form pairs by forming hydrogen bonds. "Complementary," as used herein, refers to the ability to form an exact pair between two nucleotides. For example, if a nucleotide at a particular position in an oligonucleotide can hydrogen bond to a nucleotide at the same position in a DNA or RNA molecule, the oligonucleotide and DNA or RNA are likely to be complementary to each other at that position. . Oligonucleotides and DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides capable of hydrogen bonding to each other.
[0026]
Thus, "specifically hybridizable" and "complementary" refer to a sufficient degree of complementarity, that is, precise pairing that results in stable and specific binding between the oligonucleotide and the DNA or RNA target. Is a term used to indicate Oligonucleotides are specifically hybridizable to their target sequence by forming base pairs between specific partner nucleobases within the nucleic acid duplex. Of the natural nucleobases, guanine (G) binds to cytosine (C) and adenine (A) binds to thymine (T) or uracil (U). In addition to the equivalence of U (RNA) and T (DNA) as partners of A, other natural nucleobase equivalents are known, including 5-methylcytosine and 5-hydroxymethylcytosine (HMC) ( C equivalent), as well as 5-hydroxymethyluracil (U equivalent). In addition, synthetic nucleobases that retain partner specificity are known in the art, including, for example, 7-deaza-guanine that retains partner specificity for C. Thus, the ability of an oligonucleotide to specifically hybridize to a target sequence is not altered by chemical modifications to the nucleobase in the nucleotide sequence of the oligonucleotide, which impacts its specificity for a partner nucleobase in the target nucleic acid. I will not give.
[0027]
It is understood in the art that the nucleobase sequence of an oligonucleotide or other antisense compound need not be 100% complementary to its target nucleic acid sequence in order to be specifically hybridizable. Under specific conditions where specific binding is desired, i.e., under physiological conditions for in vivo assays or therapeutic treatments, or under assay conditions for in vitro assays, the non-specificity of the oligonucleotide to the non-target sequence An antisense compound is capable of specifically hybridizing to its target nucleic acid when there is a sufficient degree of complementarity to avoid specific binding.
[0028]
Antisense oligonucleotides are commonly used as research reagents, diagnostic aids, and therapeutics. For example, antisense oligonucleotides capable of inhibiting the expression of a gene with excellent specificity are often used by those skilled in the art to elucidate the function of a particular gene, for example, by using various pathways of the biological pathway. Used to distinguish member functions. Thus, this specific inhibitory effect has been exploited by those skilled in the art for research use. The specificity and sensitivity of oligonucleotides is also utilized by those skilled in the art for therapeutic uses. Specific examples of preferred antisense compounds useful in the present invention include oligonucleotides containing modified backbones or unnatural intersugar linkages. As defined herein, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes mentioned in the art, modified oligonucleotides that do not have a phosphorus atom in the intersugar backbone can also be considered oligonucleosides.
[0029]
Specific oligonucleotide chemical modifications are described in the following subsections. Not all positions of a given compound need be uniformly modified; in fact, two or more of the following modifications can be incorporated into a single antisense compound, or a single residue thereof, for example: Even a single nucleoside within an oligonucleotide can be incorporated.
[0030]
A. Modified Ligation: Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphoro-dithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates ), Phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoraamidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and Boranophosphates having a 3'-5 'linkage, their 2'-5' linked analogs, and pairs of adjacent nucleotide units having a 3'-5 'to 5'-3' or 2'-5 'to 5'- The pole that connects to 2 ' There include those inverted. Various salts, mixed salts and free acid forms are also included.
[0031]
Representative U.S. Patents that teach the preparation of the phosphorus atom-containing linkages include U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243. No. 5,177,196; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; No. 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571. No. 5,799, 793; No. 5,587,361; Nos. 625,050; and 5,697,248, each of which is incorporated herein by reference.
[0032]
Preferred modified oligonucleotide backbones that do not contain a phosphorus atom (ie, oligonucleosides) include short chain alkyl or cycloalkyl sugar linkages, mixed heteroatoms and alkyl or cycloalkyl sugar linkages, or one or more short chains. It has a main chain formed by heteroatoms or heterocyclic sugar linkages. These include morpholino linkages (partially formed by the sugar moieties of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones. Alkylene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; those containing amide backbones; and mixed N, O, S and CH₂ Others having a component part are included.
[0033]
Representative U.S. Patents that teach the preparation of the above oligonucleosides include U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; No. 5,216,141; No. 5,235,033; No. 5,264,562; No. 5,264,564; No. 5,405,938; No. 5,434,257; 5,466,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602. 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070. No. 5,663,312; No. 5, Nos. 633,360; 5,677,437; and 5,677,439, each of which is incorporated herein by reference.
[0034]
In other preferred oligonucleotide mimetics, both the sugar and the intersugar linkage at the nucleotide unit, ie, the backbone, has been replaced with a new group. These base units are maintained for hybridization with the appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of the oligonucleotide is replaced by an amide-containing backbone, especially an aminoethylglycine backbone. These nucleobases are retained and bind directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. Patents that teach the preparation of PNA compounds include U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated by reference. (Incorporated herein), but is not limited thereto. Further teachings of PNA compounds can be found in Nielsen et al. , Science, 1991, 254, 1497.
[0035]
The most preferred embodiments of the present invention are oligonucleotides having a phosphorothioate backbone and oligonucleotides having a heteroatom backbone, particularly the -CH of U.S. Patent No. 5,489,677, referenced above.₂ -NH-O-CH₂ -, -CH₂ -N (CH₃ ) -O-CH₂ -[Known as methylene (methylimino) or MMI backbone], -CH₂ -ON (CH₃ ) -CH₂ -, -CH₂ -N (CH₃ ) -N (CH₃ ) -CH₂ -And -ON (CH₃ ) -CH₂ -CH₂ -[Where the original phosphodiester main chain is -O-P-O-CH₂ And the amide backbone of US Pat. No. 5,602,240 referenced above. Also preferred are the oligonucleotides having a morpholino backbone structure of US Pat. No. 5,034,506, referenced above.
[0036]
B. Modified nucleobases: The compounds of the present invention may further or alternatively comprise nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). . Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine. Methyl and other alkyl derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine And thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted Sills and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, include 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine. Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. Am. I. , Ed. John Wiley & Sons, 1990, Englisch et al. Angewandte Chemie, International Edition, 1991, 30, 613, and Sangvi, Y .; S. , Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S .; T. and Lebleu, B.C. , Ed. , CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, 6-azapyrimidines, and N-2, N-6 and O-6 substituted purines. 5-Methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6-1.2 ° C (previously, pages 276-278), and at this time, in particular, 2'-methoxyethyl sugars It is even more preferred when combined with modifications.
[0037]
Representative U.S. Patents that teach the preparation of certain of the above modified nucleobases in addition to other modified nucleobases include U.S. Patent No. 4,845,205 in addition to U.S. Patent No. 3,687,808 described above. No. 5,130,302; No. 5,134,066; No. 5,175,273; No. 5,367,066; No. 5,432,272; No. 5,457,187; No. 5,459,255; No. 5,484,908; No. 5,502,177; No. 5,525,711; No. 5,552,540; No. 5,587,469; Nos. 5,594,121; 5,596,091; 5,614,617; and 5,681,941, each of which is incorporated herein by reference, and December 1996. US patent application filed on the 10th 08 / 762,488 No. (also incorporated herein by reference) but are not intended to be limited thereto.
[0038]
C. Sugar modification: The antisense compounds of the present invention may additionally or alternatively include one or more substituted sugar moieties. Preferred oligonucleotides contain one of the following at the 2 'position: OH; F; O-, S-, or N-alkyl, O-, S-, or N-alkenyl, or O, S-, or N-alkynyl, wherein alkyl, alkenyl and alkynyl are substituted or unsubstituted C₁ ~ C₁₀Alkyl or C₂ ~ C₁₀Alkenyl and alkynyl). Particularly preferred are O [(CH₂ )_n O]_m CH₃ , O (CH₂ )_n OCH₃ , O (CH₂ )_n NH₂ , O (CH₂ )_n CH₃ , O (CH₂ )_n ONH₂ , And O (CH₂ )_n ON [(CH₂ )_n CH₃ )]₂ (Where n and m are from 1 to about 10). Other preferred oligonucleotides contain one of the following at the 2 'position: C₁ ~ C₁₀Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃ , OCN, Cl, Br, CN, CF₃ , OCF₃ , SOCH₃ , SO₂ CH₃ , ONO₂ , NO₂ , N₃ , NH₂ , Heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of oligonucleotide, or pharmacodynamics of oligonucleotide Groups to improve the mechanical properties, and other substituents having similar properties. Preferred modifications include 2'-methoxyethoxy [2'-O-CH₂ CH₂ OCH₃ , Aka 2'-O- (2-methoxyethyl) or 2'-MOE] (Martin et al., Helv. Chim. Acta, 1995, 78, 486), ie, alkoxyalkoxy groups. Additional preferred modifications include 2'- as described in co-owned U.S. patent application Ser. No. 09 / 016,520, filed Jan. 30, 1998, the contents of which are incorporated herein by reference. Dimethylaminooxyethoxy, that is, O (CH₂ )₂ ON (CH)_{3 2} Group, also known as 2'-DMAOE.
[0039]
Other preferred modifications include 2'-methoxy (2'-O-CH₃ ), 2'-aminopropoxy (2'-OCH₂ CH₂ CH₂ NH₂ ) And 2'-fluoro (2'-F). Similar modifications can be made at other positions on the oligonucleotide, especially the 3 'position of the sugar on the 3' terminal nucleotide or at the 2'-5 'linked nucleotide and the 5' position on the 5 'terminal nucleotide. Oligonucleotides may have sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugars. Representative U.S. Patents that teach the preparation of such modified sugar structures include U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359, No. 5,393,878; No. 5,446,137; No. 5,466,786; No. 5,514,785; No. 5,519,134; No. 5,567,811. No. 5,576,427; No. 5,591,722; No. 5,597,909; No. 5,610,300; No. 5,627,0531 No. 5,639,873; No. 5 5,658,873; 5,670,633; and 5,700,920, each of which is incorporated herein by reference, and June 1995. US Patent Application No. 0 filed on the 5th / No. 468,037 (which is also incorporated herein by reference) include.
[0040]
D. Other Modifications: Further modifications may be made at other positions on the oligonucleotide, especially the 3 'position of the sugar at the 3' terminal nucleotide and the 5 'position of the 5' nucleotide. For example, one further modification of the oligonucleotide of the invention involves chemically linking one or more moieties or conjugates to the oligonucleotide that enhances the activity, cellular distribution or cellular uptake of the oligonucleotide. . Such moieties include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bior. Med. Chem. Let., 1993, 3, 2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), aliphatic chains, e.g. For example, dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., I., I. 75, 49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium, 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., Shea et al., Nucl. Acids Res., 1990, 18, 3777), polyamines or polyethylene glycols. (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 365, 365, 365, 365, 365, 365, 365, 365, 365, 365). , 1995, 1264, 229) or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923). Not something.
[0041]
Representative U.S. Patents that teach the preparation of such oligonucleotide conjugates include U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525. 5,465,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731. No. 5,591,584; No. 5,109,124; No. 5,118,802; No. 5,138,045; No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735; No. 4,667,025; No. 4,789,7; No. 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830 No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; Nos. 371,241 and 5,391,723; 5.416,203, 5,451,463; 5,510,475; 5,512,667; 5,514. No. 785; 5,565,552; 5,567,8 No. 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923. Nos. 5,599,928 and 5,688,941, each of which is incorporated herein by reference, but is not limited thereto.
[0042]
E. FIG. Chimeric oligonucleotides: The invention also includes antisense compounds, which are chimeric compounds. In the context of the present invention, a "chimeric" antisense compound or "chimera" is an antisense comprising two or more chemically distinct regions each consisting of at least one monomer unit, ie a nucleotide in the case of an oligonucleotide. Compounds, especially oligonucleotides. These oligonucleotides typically include at least one region where the oligonucleotide is provided with increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. Is qualified. This additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA: DNA or RNA: RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA: DNA duplex. Thus, activating RNAse H results in cleavage of the RNA target, thereby greatly increasing the efficiency of oligonucleotide inhibition of gene expression. Thus, compared to phosphorothioate deoxyoligonucleotides that hybridize to the same target region, comparable results can often be obtained with shorter oligonucleotides when using chimeric oligonucleotides. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art. RNase H-mediated cleavage of the target is distinct from the use of ribozymes for cleavage of nucleic acids, and ribozymes are not included in the present invention.
[0043]
By way of example, such a "chimera" is a "gapmer", i.e., the central portion of the oligonucleotide (the "gap") serves, for example, as a substrate for RNase H, and the 5 'and 3' portions (" Wings) are modified in such a way that they have a higher affinity for the target RNA molecule, or have a higher stability if they form a duplex with it, but do not support nuclease activity. It can be a possible (eg, 2'-fluoro- or 2'-methoxyethoxy-substituted) oligonucleotide. Other chimeras include "hemimers", i.e., the 5 'portion of the oligonucleotide serves as a substrate for, for example, RNase H, whereas the 3' portion has a higher affinity for the target RNA molecule. Or when it forms a duplex with it, but is modified in such a way as to have greater stability, but is unable to support nuclease activity (eg 2'-fluoro- or 2'- Methoxyethoxy-substituted), or vice versa.
[0044]
Chemical modifications to some oligonucleotides that confer higher oligonucleotide: RNA duplex stability have been described by Freier et al. (Nucl. Acids Res., 1997, 25, 4429). Such modifications are preferred for the RNase H resistant portion of the chimeric oligonucleotide and can generally be used to increase the affinity of the antisense compound for target RNA.
The chimeric antisense compounds of the present invention can be formed as a composite structure of two or more of the above-described oligonucleotides, modified oligonucleotides, oligonucleosides and / or oligonucleotide mimetics. Such compounds are also referred to in the art as hybrids or gapmers. Representative U.S. Patents that teach the preparation of such hybrid structures include U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775. No. 5,366,878; No. 5,403,711; No. 5,491,133; No. 5,565,350; No. 5,623,065; No. 5,652,355; Nos. 5,652,356; and 5,700,922, each of which is incorporated herein by reference, and US patent application Ser. No. 08 / 465,880, filed Jun. 6, 1995. (Also incorporated herein by reference), but is not limited thereto.
[0045]
F. Synthesis: The oligonucleotides used in accordance with the present invention can be conveniently and routinely made by known solid-phase synthesis techniques. Equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis known in the art may additionally or alternatively be used. It is also known to use similar techniques for the preparation of other oligonucleotides, such as phosphorothioates and alkylated derivatives.
[0046]
1. Techniques for the synthesis of certain modified oligonucleotides can be found in the following U.S. Patents or continuing patent applications: U.S. Patent Nos. 5,138,045 and 5,218,105 for polyamine-linked oligonucleotides; U.S. Pat. No. 5,212,295 for monomers to prepare oligonucleotides having a modified backbone; U.S. Pat. Nos. 5,378,825 and 5,541,307 for oligonucleotides having a modified backbone; U.S. Pat. No. 5,386,023 for strand modified oligonucleotides and their preparation by reductive coupling; U.S. Pat. No. 5,457,191 for modified nucleobases based on the 3-deazapurine ring system and methods for their synthesis. A modified nucleobase based on an N-2-substituted purine U.S. Pat. No. 5,459,255; U.S. Pat. No. 5,521,302 for methods of preparing oligonucleotides having a chiral phosphorus linkage; U.S. Pat. No. 5,539,082 for peptide nucleic acids; .beta.-lactam backbone US Pat. No. 5,554,746 for oligonucleotides having the formula: US Pat. No. 5,571,902 for methods and materials for synthesizing oligonucleotides; nucleosides having an alkylthio group (such groups are known as nucleotides). U.S. Pat. No. 5,578,718; for oligonucleotides having high chiral purity phosphorothioate linkages, U.S. Pat. 587,361 and 5,599,797; No. 5,506,351 for methods of preparing '-O-alkylguanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. U.S. Patent No. 5,587,470 for oligonucleotides having 3-deazapurine; U.S. Patent No. 5,223, issued June 29, 1993 for conjugated 4'-desmethylnucleoside analogs. U.S. Patent Nos. 5,602,240 and 5,610,289 for backbone modified oligonucleotide analogs; and, inter alia, 2'-fluoro. -Regarding the method of synthesizing oligonucleotides, see U.S. Patent Application No. 08/38, filed February 3, 1995. 3,666, and U.S. Patent No. 5,459,255.
[0047]
2. Bioequivalents: The compounds of the present invention can be any pharmaceutically acceptable salt, ester, or salt of such an ester, or a biologically active metabolite or a metabolite when administered to animals, including humans. Include any other compounds that can provide (directly or indirectly) those residues. Thus, for example, this disclosure also discloses "prodrugs" and "pharmaceutically acceptable salts" of antisense compounds of the present invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. About things.
[0048]
A. Oligonucleotide prodrugs: The antisense compounds of the present invention can additionally or alternatively be prepared to be delivered in "prodrug" form. A “prodrug” is a therapeutic agent prepared in an inactive form, which is converted into an active form (ie, drug) by endogenous enzymes or other chemicals and / or conditions in the body or their cells. Show. In particular, prodrug versions of the antisense compounds of the present invention can be prepared using the SATE [(S-acetyl-2-thioethyl) method according to the method disclosed in Gosselin et al., WO 93/24510 published December 9, 1993. Phosphate] derivatives.
[0049]
B. Pharmaceutically acceptable salts: The term "pharmaceutically acceptable salts" refers to the physiologically and pharmaceutically acceptable salts of antisense compounds of the invention: that is, to retain the desired biological activity of the parent compound. And does not impart to them undesirable toxicological effects.
[0050]
Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium and the like. Examples of suitable amines are chloroprocaine, choline, N, N'-dibenzylethylenediamine, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts, J. of Pharma Sci., 1977, 66: 1). The base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the usual manner. The free acid form can be regenerated by bringing the salt form into contact with an acid and isolating the free acid in the usual manner. The free acid forms differ somewhat from their respective salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are different from their respective free acids for the purposes of the present invention. equal. As used herein, “pharmaceutical addition salts” include pharmaceutically acceptable salts of one of the components of the compositions of the invention in the acid form. These include organic or inorganic acid salts of amines. Preferred acid salts are hydrochloride, acetate, salicylate, nitrate and phosphate. Other suitable pharmaceutically acceptable salts are known to those skilled in the art, and include basic salts of various inorganic and organic acids, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, or phosphorous. Acids; organic carboxylic acids, sulfonic acids, sulfo or phospho acids or N-substituted sulfamic acids such as acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, Tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, embonic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, nicotinic acid, isonicotine Acids or 2-acetoxybenzoic acids; and amino acids, such as the 20 alpha-amino acids involved in protein synthesis in nature, e.g. For example, glutamic acid or aspartic acid, and further, naphthalene-1,5-disulfonic acid, phenylacetic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, methanesulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with formation of cyclamate), or other acidic organic compounds; For example, ascorbic acid is included. Pharmaceutically acceptable salts of the compounds can also be prepared using pharmaceutically acceptable cations. Suitable pharmaceutically acceptable cations are known to those of skill in the art, and include alkali, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or carbonic acids are also possible.
[0051]
For oligonucleotides, preferred examples of pharmaceutically acceptable salts include (a) salts formed with cations, such as sodium, potassium, ammonium, magnesium, calcium, polyamines, such as spermine and spermidine; b) an acid addition salt formed with an organic acid, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid or the like; (c) an organic acid, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, Fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid And (d) anions in elemental state, for example, chlorine, bromine And including but salts formed with iodine, but is not limited thereto.
[0052]
3. Exemplary Utility of the Invention: The oligonucleotides of the invention hybridize specifically to nucleic acids (eg, mRNA) encoding LFA-3 protein. The antisense compounds of the present invention can be utilized as therapeutic compounds, diagnostic tools or research reagents that can be incorporated into other methodologies in addition to kits, as will be apparent to those skilled in the art.
[0053]
A. Assays and diagnostic applications: The oligonucleotides of the invention can be used to detect the presence of LFA-3 protein-specific nucleic acids in cell or tissue samples. For example, using a polynucleotide kinase,³²Radiolabeled oligonucleotides can be prepared by P-labeling. (Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2, pg. 10.59). Next, the radiolabeled oligonucleotide is contacted with a cell or tissue sample suspected of containing LFA-3 protein messenger RNA (and thus LFA-3 protein) and washed to remove unbound oligonucleotide. The radioactivity remaining in the sample indicates the presence of the bound oligonucleotide, which in turn indicates the presence of a nucleic acid complementary to the oligonucleotide, using a scintillation counter or other routine means. Can be quantified. In this way, the expression of nucleic acids encoding these proteins is detected.
[0054]
The radiolabeled oligonucleotides of the present invention can also be used to perform autoradiography on tissue to determine the localization, distribution and quantification of LFA-3 protein for research, diagnostic or therapeutic purposes. In such studies, tissue sections are treated with a radiolabeled oligonucleotide, washed as described above, and then exposed to a photographic emulsion according to routine autoradiographic procedures. This emulsion, when developed, produces an image of silver particles throughout the region expressing the LFA-3 protein gene. Quantitation of silver particles allows for the detection of expression of mRNA molecules encoding these proteins, and allows targeting of oligonucleotides to these regions.
[0055]
Similar assays can be developed for fluorescent detection of LFA-3 protein nucleic acid expression using oligonucleotides of the invention conjugated to fluorescein or other fluorescent tags instead of radiolabels. Such conjugates are routinely achieved during solid-phase synthesis using fluorescently labeled amidite or controlled pore glass (CPG) columns. Fluorescein-labelled amidites and CPGs are available, for example, from Glen Research, Sterling VA. Other means of labeling oligonucleotides are known in the art (e.g., Ruth, Chapter 6 In: Methods in Molecular Biology, Vol. 26: Protocols for Oligonucleotides, Australia, Agra. , NJ, 1994, pages 167-185).
[0056]
Kits for detecting the presence or absence of LFA-3 protein can also be prepared. Such kits include oligonucleotides targeted to the appropriate gene, ie, the gene encoding the LFA-3 protein. Suitable kits and assay formats, eg, "sandwich" assays, are known in the art and can be readily adapted for use with the antisense compounds of the present invention. Hybridization between the antisense compound of the present invention and a nucleic acid encoding an LFA-3 protein can be detected by means known in the art. Such means may include conjugation of the enzyme to the oligonucleotide, radiolabeling of the oligonucleotide or any other suitable detection system.
[0057]
B. Biologically active oligonucleotides: The present invention also relates to the administration of biologically active oligonucleotides to cultured cells, isolated tissues and organs, and animals. By "biologically active" is meant that the oligonucleotide functions to regulate the expression of one or more genes in cultured cells, isolated tissues or organs and / or animals. Such regulation can be achieved by antisense oligonucleotides by various mechanisms known in the art, including transcription termination; effects on RNA processing (copy, polyadenylation and splicing) and transport; target nucleic acids And translational arrest (Crooke et al., Exp. Opin. Ther. Patents, 1996, 6: 855).
[0058]
In non-human animals, the compositions and methods of the invention can be used to study the function of one or more genes in the animal. For example, antisense oligonucleotides have been used in rats to study the role of N-methyl-D-aspartate receptors in neuronal death and in mice to study the biological role of protein kinase C-α. And systemic administration to rats to examine the role of the neuropeptide Y1 receptor in anxiety (Wahlestedt et al., Nature, 1993, 363: 260, respectively; Dean et al., Proc. Natl. Acad. Scad. U.S.A., 1994, 91: 11762; and Wahlestedt et al., Science, 1993, 259: 528). When a complex family of related proteins is being studied, "antisense knockout" (ie, inhibition of a gene by systemic administration of an antisense oligonucleotide) is the most accurate means to determine a particular member of that family. (Generally see Albert et al., Trends Pharmacol. Sci., 1994, 15: 250).
[0059]
The compositions and methods of the invention have (or are afflicted with) or are prone to have a disease or disorder that is wholly or partially treatable with one or more nucleic acids. There are also therapeutic uses in animals, including humans, which are known or suspected. The term "therapeutic use" is intended to include prophylactic, palliative and therapeutic uses where the antisense compounds of the present invention are contacted with the cells of the animal, either in vivo or ex vivo. When brought into contact with animal cells ex vivo, therapeutic uses include incorporating such cells into animals after treatment with one or more antisense compounds of the present invention.
[0060]
For therapeutic use, animals suspected of having a disease or disorder that can be treated or prevented by modulating LFA-3 expression or activity are treated, for example, by administering an oligonucleotide according to the present invention. . The antisense compounds of the present invention can be utilized in pharmaceutical compositions by adding an effective amount of the oligonucleotide to a suitable pharmaceutically acceptable carrier, such as a diluent. One of skill in the art has identified antisense, triplex and other oligonucleotide compositions that are capable of regulating the expression of genes involved in viral, fungal and metabolic diseases. Antisense oligonucleotides have been safely administered to humans, and several clinical trials are ongoing. It is thus established that oligonucleotides may be useful therapeutic tools that can be configured to be useful in therapeutic forms for treating cells, tissues and animals, especially humans. The following U.S. patents show temporary palliative, therapeutic and other methods utilizing antisense oligonucleotides. U.S. Patent No. 5,135,917 provides antisense oligonucleotides that inhibit expression of the human interleukin-1 receptor. U.S. Pat. No. 5,098,890 is directed to antisense oligonucleotides complementary to the c-myb oncogene and to antisense oligonucleotide treatment of certain cancerous conditions. U.S. Pat. No. 5,087,617 provides a method of treating cancer patients using antisense oligonucleotides. U.S. Patent No. 5,166,195 provides oligonucleotide inhibitors of human immunodeficiency virus (HIV). U.S. Patent No. 5,004,810 provides oligomers that can hybridize to herpes simplex virus Vmw65 mRNA and inhibit replication. U.S. Patent No. 5,194,428 provides antisense oligonucleotides having antiviral activity against influenza virus. U.S. Pat. No. 4,806,463 provides antisense oligonucleotides and methods of using them to inhibit HTLV-III replication. U.S. Pat. No. 5,286,717 provides an oligonucleotide having a nucleotide sequence complementary to a part of an oncogene. U.S. Patent Nos. 5,276,019 and 5,264,423 are directed to phosphorothioate oligonucleotide analogs used to prevent the replication of foreign nucleic acids in cells. U.S. Patent No. 4,689,320 is directed to antisense oligonucleotides as antiviral agents specific for cytomegalovirus (CMV). U.S. Pat. No. 5,098,890 provides oligonucleotides complementary to at least a portion of the mRNA transcript of the human c-myb gene. U.S. Patent No. 5,242,906 provides antisense oligonucleotides useful in treating latent Epstein-Barr virus (EBV) infection.
[0061]
As used herein, the term “disease or disorder” refers to (1) any abnormal or infective nature of an organism or part, particularly as a result of infection, innate weakness, environmental stress that impairs normal physiological function. (2) excludes pregnancy itself except for autoimmunity and other diseases associated with pregnancy; and (3) includes cancer and tumors. The term “known or suspected of having a propensity to have a disease or disorder” is defined herein that a subject has a high risk to the general population of such animals. Indicates that a particular disease or disorder has been determined to occur or is suspected. For example, a subject animal that is "known or suspected of having a disease or disorder" may have a personal and / or family medical history that includes frequent occurrences of a particular disease or disorder. is there. As another example, a subject "known to be, or suspected of having, a disease or disorder" is susceptible to susceptibility as determined by genetic screening according to techniques known in the art. (E.g., US Congress, Office of Technology Assessment, Chapter 5 In; Genetic Monitoring and Screening in the Workplace, OTA-BA-455-G.U. C., 1990, pages 75-99). The term “disease or disorder that is wholly or partially treatable with one or more antisense compounds” refers to a disease or disorder as defined herein, including (1) their management, modulation or treatment, And / or (2) therapeutic, curative, palliative and / or prophylactic relief from them may be provided by administering a composition comprising one or more antisense compounds of the present invention. .
[0062]
4. Pharmaceutical Compositions Containing the Compounds of the Invention: The present invention has provisions for therapeutic and pharmaceutical compositions comprising one or more LFA-3-modulating antisense compounds. Compositions for administering the antisense compounds of the present invention include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
[0063]
A. Composition for Dietary Delivery: In a preferred embodiment of the invention, one or more LFA-3 modulating antisense compounds are administered by dietary delivery, preferably by oral administration. Pharmaceutical compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, troches, tablets or SECs (soft elastic capsules or "caplets"). Thickeners, flavorings, diluents, emulsifiers, dispersing aids, carrier substances or binders can be added to such compositions. Such pharmaceutical compositions have the effect of delivering the antisense compound (s) to the gastrointestinal tract for exposure to their mucous membranes. Thus, the pharmaceutical composition should include substances that are effective in protecting the oligonucleotides from gastric pH flow or releasing the oligonucleotides over time to optimize their delivery to specific mucosal sites. Can be. Enteric coatings for acid-resistant tablets, capsules and caplets are known in the art, and typically include acetate phthalate, polypropylene glycol and sorbitan monooleate. Various methods are known in the art for preparing pharmaceutical compositions for dietary delivery. Generally, Remington's Pharmaceutical Sciences, 18th Ed. , Gennaro, ed. . Mac Publishing Co., Ltd. See, Nairn, Chapter 83; Block, 87; Rudnic et al., 89; Porter, Chapter 90; and Longer et al., Chapter 91, Easton, PA, 1990.
[0064]
The antisense compounds of the present invention are inert and non-toxic pharmaceutically acceptable in a known manner in conventional pharmaceutical compositions such as tablets, coated tablets, pills, granules, aerosols, syrups, emulsions, suspensions and solutions. It can be incorporated with a possible carrier (excipient). The therapeutically active compound should in each case be present in a concentration of from about 0.5% to about 95% by weight of the total mixture, i.e. in an amount sufficient to achieve the stated dosage range. is there. The pharmaceutical composition is prepared, for example, by diluting the active compound with a pharmaceutically acceptable carrier and, if appropriate, using emulsifiers and / or dispersants, wherein water is used as the diluent. Sometimes, where appropriate, organic solvents can be used as auxiliaries. The pharmaceutical composition can, if appropriate, be formulated in a conventional manner using additional pharmaceutically acceptable carriers. Thus, these compositions may contain, by conventional means, additional excipients, for example, a binder (eg, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); a filler (eg, lactose, finely divided). Prepared using a lubricant (eg, magnesium stearate, talc or silica); a disintegrant (eg, starch or sodium starch glycolate); or a wetting agent (eg, sodium lauryl sulfate). can do. Tablets may be coated by methods known in the art and contain flavoring, coloring and / or sweetening agents.
[0065]
Compositions comprising one or more LFA-3 modulating antisense compounds can be administered in a rectal mode. In particular, therapeutic or pharmaceutical compositions for rectal administration include foams, solutions (enemas) and suppositories. A rectal suppository for an adult usually has a tapered shape at one or both ends, and when a usual base, cacao butter is used, typically weighs about 2 g. It is typically about half the weight (Block 87 of Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed. Mac Publishing Co., Easton, PA, 1990, Chapter 87).
[0066]
Pharmaceutical compositions that can be conveniently presented in pharmaceutical form can be prepared according to conventional techniques known in the pharmaceutical arts. Such techniques include the step of bringing into association the active ingredient (s) with the pharmaceutically acceptable carrier (s). In general, the pharmaceutical compositions will uniformly and intimately associate the active ingredient (s) with liquid excipients or finely divided solid excipients or both, and then, if necessary, Prepared by molding the product.
[0067]
Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units, for example, capsules, cashews or tablets, each of which contains a predetermined amount of the active ingredient; as a powder or granules; As a solution or suspension in a non-aqueous liquid; or as an oil-in-water emulsion or a water-in-oil liquid emulsion. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may contain the active ingredient in free flowing form, such as a powder or granules, optionally in a suitable device, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersant. It can be prepared by compression. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets may be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient. Enteric coatings for sustained release oral delivery systems and / or oral dosage forms are disclosed in U.S. Patent Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309, No. 404.
[0068]
B. Additives: Pharmaceutical and therapeutic compositions comprising one or more antisense compounds of the present invention include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic carrier materials suitable for non-parenteral administration that do not deleteriously react with antisense compounds can be used. The pharmaceutical composition is sterilized and, if necessary, auxiliaries which do not react harmfully with the oligonucleotide (s) of the pharmaceutical composition, such as lubricants, preservatives , Stabilizers, wetting agents, emulsifiers, salts which affect osmotic pressure, buffers, coloring, flavoring and / or aroma substances and the like. Pharmaceutical compositions in the form of an aqueous suspension can include substances that increase the viscosity of the suspension, including, for example, sodium carboxymethyl cellulose, sorbitol, and / or dextran. In some cases, such compositions may include one or more stabilizers, penetration enhancers, carrier compounds or pharmaceutically acceptable carriers.
[0069]
(1) Penetration enhancers: Pharmaceutical compositions containing the oligonucleotides of the present invention may contain penetration enhancers to enhance the dietary delivery of those oligonucleotides. Penetration enhancers can be classified as belonging to one of five broad categories: fatty acids, bile salts, chelators, surfactants and non-surfactants (Lee et al., Critical Reviews in). Therapeutic Drug Carrier Systems, 1991, 8: 91-192; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7: 1).
[0070]
Fatty acids: Various fatty acids and their derivatives that act as penetration enhancers include, for example, oleic acid, lauric acid, linoleic acid, linolenic acid, dicaplate, tricaplate, recinleate, monoolein (ak. 1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arichidonic acid, glyceryl 1-monocaprate, acylcarnitine, acylcholine, 1-dodecylazacycloheptan-2-one, mono- and Includes di-glycerin and their physiologically acceptable salts (ie, oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al., Critical Revie). W. in in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical.Reviews in Therapeutic Drugs, Pharmaceuticals, Inc., 1992;
[0071]
Bile salts: Physiological roles of bile include the promotion of dispersion and absorption of lipids and fat-soluble vitamins (Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., Med., Ed., Ed. Hill, New York, NY, 1996, pages 934-935, Brunton, Chapter 38). Various natural bile salts, and their synthetic derivatives, act as osmotic vehicles. Thus, "bile salts" include any of the natural components of bile and any of their synthetic derivatives.
[0072]
Chelating agents: Chelating agents have the further advantage of also acting as a DNase inhibitor, including citric acid, disodium ethylenediaminetetraacetate (EDTA), salicylates (eg, salicylic acid, 5-methoxysalicylic acid and homovanillic acid) Sodium), N-acyl derivatives of collagen, laureth-9, and N-aminoacyl derivatives of beta-diketone (enamine), but are not limited thereto (Lee et al., Crit). Rev. Therap.Drug Carrier Systems, 1991, p.92; Muranishi, Crit.Rev.Therap.Drug Carrier Systems, 1990, 7, 1; Bur et al. rol Rel., 1990, 14, 43).
[0073]
Surfactants: Examples of surfactants include sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page). 92); and perfluorochemical emulsions such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988, 40: 252).
[0074]
Non-surfactants: Non-surfactants include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92). And non-steroidal anti-inflammatory agents such as diclofenac sodium, iodomethasin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39: 621).
[0075]
(2) Carrier compound: As used herein, a “carrier compound” is a nucleic acid or an analog thereof that is inactive (ie, has no biological activity itself) but is biologically active. Recognized as a nucleic acid by an in vivo process that reduces the bioavailability of an active nucleic acid, for example, by degrading the biologically active nucleic acid or promoting its removal from the circulation Refers to something. Co-administration of the nucleic acid and the carrier compound, typically in excess of the latter substance, may result in liver, kidney or other circulating, possibly due to competition between the carrier compound and the nucleic acid for a common receptor. A substantial reduction in the amount of nucleic acid recovered in the external reservoir can occur. For example, recovery of a partially phosphorothioated oligonucleotide in liver tissue indicates that it is polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfone. Decreases when co-administered with acid (Miyao et al., Antisense Res. Dev., 1995, 5: 115; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 1: 1, 1996).
[0076]
(3) Pharmaceutically acceptable carrier: In contrast to a carrier compound, a "pharmaceutically acceptable carrier" (excipient) is a pharmaceutically acceptable carrier for delivering one or more nucleic acids to an animal. It is an acceptable solvent, suspending agent or any other pharmacologically inert vehicle. The pharmaceutically acceptable carrier may be liquid or solid and, when combined with the nucleic acid and other components of the given pharmaceutical composition, is administered in a manner envisioned to provide the desired bulk, content, etc. Selected according to the method. Typical pharmaceutically acceptable carriers include binders such as pre-gelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose; fillers such as lactose and other sugars, microcrystalline cellulose, Pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate or calcium hydrogen phosphate, etc .; lubricants (eg, magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearate, hydrogenated vegetable oil, corn starch, Disintegrants (e.g., starch, sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Not. Suitable pharmaceutically acceptable carriers include water, saline, alcohol, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, hydroxymethylcellulose, polyvinylpyrrolidone viscous paraffin and the like. However, the present invention is not limited to these.
[0077]
(4) Various further ingredients: The compositions of the present invention may further comprise other auxiliary ingredients conventionally found in pharmaceutical compositions at their art-established use levels. Thus, for example, these compositions may contain additional compatible pharmaceutically active substances, such as antipruritics, astringents, local anesthetics or anti-inflammatory agents, or various compositions of the present invention. Additional substances useful in physically formulating the dosage form can be included such as dyes, flavors, preservatives, antioxidants, opacifiers, thickeners and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
[0078]
C. Colloidal dispersions: Regardless of the method by which the antisense compounds of the present invention are introduced into a patient, colloidal dispersions are used as delivery vehicles to enhance the in vivo stability of those compounds and / or identify those compounds. Target organs, tissues or cell types. Colloidal dispersions include macromolecular complexes, nanocapsules, microspheres, beads, and oil-in-water emulsions, micelles, mixed micelles, lipid-based systems including uncharacterized lipid: oligonucleotide complexes and liposomes. But are not limited to these.
[0079]
A preferred colloidal dispersion is a plurality of liposomes. Liposomes are microscopic spheres with an aqueous nucleus surrounded by one or more outer layer (s) consisting of lipids arranged in a bilayer configuration (generally, Chonn et al., Current Op. Biotech., 1995, 6, 698). The therapeutic potential of liposomes as drug delivery agents was recognized almost 30 years ago (Sessa et al., J. Lipid Res., 1968, 9, 310). Liposomes can be used in some cases, as a cellular delivery vehicle for bioactive agents, in vitro and in vivo (Mannino et al., Biotechniques, 1988, 6,682; Blume et al.). , Biochem. Et Biophys. Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119). For example, it has been shown that macromonolayer vehicles (LUVs) ranging in size from 0.2-0.4 microns can encapsulate a significant percentage of aqueous buffers containing large macromolecules. RNA, DNA and native virions can be encapsulated in an aqueous interior and delivered to brain cells in a biologically active form (Fraley et al, Trends Biochem. Sci., 1981, 6, 77).
[0080]
The targeting of colloidal dispersions, including liposomes, can be either passive or active. Passive targeting takes advantage of the natural tendency of liposomes to distribute to cells of the reticuloendothelial system of organs including sinusoids. In contrast, active targeting involves the modification of liposomes, which is described in the viral protein coat (Morishita et al., Proc. Natl. Acad. Sci. (U.S.A.), 1993, 90, 8474), by binding a specific ligand such as a monoclonal antibody (or a suitable binding portion thereof), a sugar, a glycolipid or a protein (or a suitable oligopeptide fragment thereof), or other than at the natural localization site By changing the composition and / or size of the liposomes to achieve distribution of the liposomes into organs and cell types. The surface of the targeted colloidal dispersion can be modified in various ways. In the case of liposome-targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of the liposome to maintain the targeting ligand in tight association with the lipid bilayer. Various linking groups can be used to attach the lipid chain to the targeting ligand. This targeting ligand that binds to a particular cell surface molecule that is widely found on the cells where it is desired to deliver the compound of the present invention may be, for example, (1) a particular cell that is predominantly expressed by the cell for which delivery is desired. Hormones, growth factors or suitable oligopeptide fragments thereof, or (2) polyclonal or monoclonal antibodies that specifically bind to antigenic epitopes widely found on target cells, or their appropriate, bound by receptors Fragments (eg, Fab; F (ab '))₂ ). Two or more bioactive agents (eg, an antisense oligonucleotide and a conventional drug; two oligonucleotides) can be combined and delivered in a single liposome. Agents that enhance the intracellular stability and / or targeting of their contents can also be added to the colloidal dispersions.
[0081]
The liposomes of the present invention generally comprise a small lipid comprising one or more neutral or negatively charged phospholipids, preferably one or more neutral phospholipids, usually in combination with one or more sterols, preferably cholesterol. It is formed from vesicle-forming lipids. Examples of lipids useful for liposome formation include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, sphingolipids, phosphatidylethanolamine, cerebroside and gangliosides. Typically, the tumor lipid component of the liposome is phosphatidylcholine (PC) or a PC derivative. PC derivatives having various acyl chain groups with various chain lengths and degrees of saturation are commercially available or can be synthesized by known techniques. Less sterile PCs are generally easier to size for filtration sterilization, especially if the liposomes must be less than about 0.3 microns in size. C₁₄~ C₂₂, Especially C₁₆~ C₁₈PCs containing saturated fatty acids having a carbon chain length in the range of 1 to 2 are preferred, especially diacylphosphatidylglycerol. Illustrative phospholipids include, for example, dipalmitoyl phosphatidylcholine, phosphatidylcholine and disteroylphosphatidylcholine. Phosphatidylcholines having mono- and di-unsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used. Other suitable phospholipids include those with head groups other than choline, for example, ethanolamine, serine, glycerol, and inositol. Other suitable lipids include phosphonolipids in which the fatty acid is linked to glycerol in an ether linkage rather than an ester linkage. Preferred liposomes comprise sterols, for example, cholesterol, in a molar ratio of about 0.1 to 1.0 (sterol: phospholipid).
[0082]
Typically, the liposomes of the present invention contain about 0.005 ng / mL to about 400 mg / mL, preferably about 0.01 ng / mL to about 200 mg, of one or more antisense oligonucleotides in their aqueous interior. / Ml, most preferably from about 0.1 ng / mL to about 100 mg / mL, where "about" indicates ± 5% of the desired concentration.
[0083]
The compositions of the present invention can include one or more antisense compounds and / or other therapeutic agents entrapped in sterically stable liposomes. As used herein, the term "sterically stable liposome" refers to a liposome containing one or more specialized lipids, wherein the specialized lipid is Refers to those that result in enhanced circulatory life for liposomes lacking specialized lipids. An example of a sterically stabilized liposome is that the vesicle-forming lipid portion of the liposome is (A) one or more glycolipids, for example, monosialoganglioside G_M1Or (B) derivatized with one or more hydrophilic polymers, for example, polyethylene glycol (PEG) moieties. Although not wishing to be bound by any particular theory, the art is not concerned with at least sterically stable liposomes containing gangliosides, sphingomyelin, or PEG-derived lipids. It is believed that the enhanced circulatory half-life results from reduced cellular uptake of the reticuloendothelial system (Allen et al., FEBS Letts., 1987, 223, 42; Wu et al., Cancer Res., 1993, 53, 3765).
[0084]
Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) describe a monosilanoganglioside G that improves the blood half-life of liposomes._M1Reported the ability of galactocerebroside sulfate and phosphatidylinositol. These findings were described by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). Allen et al., U.S. Pat. Nos. 4,837,028 and WO 88/04924, describe (1) sphingomyelin and (2) ganglioside G._M1Alternatively, a liposome containing galactocerebroside sulfate is disclosed. U.S. Patent 5,543,152 (Webb et al.) Discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoyl phosphatidylcholine are disclosed in WO 97/13499 (Lim et al.).
[0085]
Many liposomes containing lipids derivatized with one or more hydrophilic polymers and methods for their preparation are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) describe a nonionic detergent containing a PEG moiety, 2C.₁₂Liposomes containing 15G have been described. (FEBS Letters, 1984, 167, 79) note that hydrophilic coating of polystyrene particles with polymeric glycols results in significant enhancement of blood half-life. Synthetic phospholipids that have been modified by the attachment of a carboxyl group of a polyalkylene glycol (eg, PEG) have been described by Sears (US Pat. Nos. 4,426,330 and 4,534,899). (FEBS Letts., 1990, 268, 235) conducted experiments showing that liposomes containing phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significantly increased the blood circulation half-life. Has been described. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) have made such observations on other PEG-derived phospholipids, such as DSPE-PEG formed from a combination of distearoyl phosphatidylethanolamine (DSPE) and PEG. Expanded. Liposomes with a PEG moiety covalently attached to the outer surface are described in Fisher's European Patents 0,445,131 B1 and WO 90/04384. Liposomal compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of using them, are described in Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin. (US Pat. No. 5,213,804 and European Patent EP 0,496,813 B1). Liposomes containing some other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Patent No. 5,225,212 (both Martin et al.) And WO 94/20073 (Zalippsky et al.). Liposomes containing PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.). U.S. Patent Nos. 5,540,935 (Miyazaki et al.) And 5,556,948 (Tagawa et al.) Describe PEG-containing liposomes that can be further derivatized with functional surface moieties.
[0086]
Liposomes containing a limited number of nucleic acids are known in the art. Published PCT application WO 96/40062 by Thierry et al. Discloses a method for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. Discloses protein-bound liposomes, claiming that the contents of such liposomes can include antisense RNA. Rahman et al., US Pat. No. 5,665,710, describes a method for encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. Discloses liposomes containing antisense oligonucleotides targeted towards the raf gene. WO 97/46671 to Klimuk et al. Discloses liposomes containing antisense oligonucleotides targeted to the gene encoding ICAM-1. One or more antisense compounds of the present invention may comprise one or more cationic lipids as disclosed in US Pat. No. 5,705,385 to Bally et al. And WO 96/40964 to Wheeler et al. (Antisense Compounds)], or as described in Kim et al., WO 98/00556.
[0087]
The liposomes of the present invention can be prepared by any of a variety of known techniques. For example, liposomes can be prepared by any conventional technique for preparing multilamellar lipid vesicles (MLVs), i.e., dissolving one or more selected lipids on the inner wall of a suitable vessel, dissolving the lipids in chloroform, After evaporating the chloroform, an aqueous solution containing the drug (s) to be encapsulated is added to the container, the aqueous solution is hydrated with lipids, and the resulting lipid suspension is swirled or vortexed. It can be formed by depositing by stirring. This process results in a mixture containing the desired liposomes.
[0088]
As another example, techniques used for the production of large unilamellar vesicles (LUV), such as reverse phase evaporation, injection methods and detergent dilution, can be used for the production of liposomes. These and other methods for generating lipid vesicles are described in Liposome Technology, Volume I (Gregoridis, Ed., CRC Press, Boca Raton, FL, 1984). These liposomes are described in U.S. Pat. Nos. 4,588,578 and 4,610,868 (both Fountain et al.), 4,522,803 (Lenk et al.) And 5,008,050 (Cullis). Steroid lipid vesicles, stable multilamellar vesicles (SPLV), monophasic vesicles (MPV) or lipid matrix carriers (LMC) of the type disclosed in US Pat. In the case of MLV, if desired, subject the liposomes to multiple (5 or more) freeze-thaw cycles to increase their trapped volume and trapping efficiency, and to obtain a more uniform intraphase distribution of solutes. (Mayer et al., J. Biol. Chem., 1985, 260, 802). Specific methods for making specific oligodeoxynucleotide: liposome compositions are described in Rahman et al., US Pat. No. 5,665,710.
[0089]
Following their preparation, the liposomes can be sized to achieve the desired size range and relatively narrow distribution of sized particles. In a preferred embodiment, the liposome has a lower range of about 50 to about 75 nM, most preferably about 60 nM in diameter, and an upper range of about 75 to about 150 nM, most preferably about 125 nM, wherein "about" ± 10 nM is shown.
[0090]
Several techniques can be used to size the liposomes into the desired size range. Sonication of the liposome suspension, either by bath or probe sonication, results in a progressive size reduction down to small unilamellar vesicles (SUVs) less than about 0.05 microns in size . Homogenization, which relies on shear energy to fragment large liposomes into smaller ones, is another known sizing technique, in which MLVs are used to reduce the size range of the selected liposomes, typically from about 0.1 to about Recirculate through a standard emulsion homogenizer until 0.5 micron is achieved. Extrusion of liposomes through a filter or membrane is another method for producing liposomes having a desired size range (see, for example, Martin et al., US Pat. No. 4,737,323 and Cullis et al., 5,008, 050). Other useful sizing methods are known to those skilled in the art. In most such methods, the particle size distribution can be monitored by conventional laser beam sizing or other means known in the art.
[0091]
Liposomes can be dehydrated using a standard lyophilizer, preferably under reduced pressure, to extend the shelf life. In an attempt to dehydrate, first the liposomes and the medium surrounding them can be frozen in liquid nitrogen and placed under reduced pressure. The addition of this latter freezing step helps prolong the overall dehydration process, but when liposomes are frozen prior to dehydration, less damage to lipid vesicles and less loss of their internal contents.
[0092]
To ensure that a significant portion of the liposomes survive this dehydration process intact, one or more protective sugars are made available for interaction with the lipid vesicle membrane and the water is removed when water is removed. Can be kept intact. Suitable sugars include, but are not limited to, trehalose, maltose, sucrose, lactose, glucose, dextran, and the like. In general, disaccharides perform better than monosaccharides and trehalose and sucrose are particularly effective in most cases, but other more complex sugars can be used instead. One skilled in the art can readily test various sugars and concentrations to determine what conditions work best for the preparation of a particular lipid vesicle (see generally Harrigan et al., Chem. Phys. Lipids, 1990, 52, 139, and Janoff et al., U.S. Patent No. 4,880,635). Generally, it has been found that a sugar concentration of about 100 mM or more produces a desired degree of protection. Once the liposomes are dehydrated, they can be stored for a long time until they are used. Suitable storage conditions depend on the chemical composition of the lipid vesicles and the active agent (s) they encapsulate. For example, liposomes containing a thermolabile drug should be stored under refrigerated conditions so that the efficacy of the active drug is not lost.
[0093]
Two or more bioactive agents (eg, oligonucleotides and conventional drugs, or two or more oligonucleotides; see below) can be combined and delivered in a single liposome. Agents that enhance the intracellular stability and / or targeting of their contents can also be added to the colloidal dispersions.
[0094]
5. Methods of Administration of the Compounds of the Invention: It is believed that the administration of therapeutic or pharmaceutical compositions containing the antisense compounds of the invention is within the skill of the art. Generally, in a patient in need of treatment or prevention, a composition comprising one or more antisense compounds according to the present invention, usually in a pharmaceutically acceptable carrier, is administered to the patient's age and the disorder or disease state to be treated. Is administered in a dose range of 0.01 μg to 100 g per kg body weight, depending on the severity of Dosing will depend on the severity and responsiveness of the disease state to be treated over the course of treatment, which lasts days to months, or until cure is achieved or reduction or prevention of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug retention in the patient's body. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimal dosages may vary depending on the relative potency of the particular antisense compound, and are generally found to be effective in in vitro and in vivo models.₅₀Can be estimated based on
[0095]
A. Therapeutic regimen: In the context of the present invention, the term "therapeutic regimen" refers to the formula for the therapeutic, palliative and prophylactic administration of one or more compositions comprising one or more antisense compounds of the present invention. It is intended to be included. A particular treatment regimen may be sustained over a period that varies depending on the nature of the particular disease or disorder, its severity and the patient's overall condition, and from once a day to every 20 years. It can be spread only once. Following treatment, the patient is monitored for changes in his / her status and for reduction of symptoms of the disorder or disease state. The dosage of the oligonucleotide can be increased if the patient does not respond significantly to the current dosage level, or if a reduction in symptoms of the disorder or disease state is observed, or the disorder or disease state If is removed, the dose can be reduced.
[0096]
Optimal dosing schedules are used to deliver a therapeutically effective amount of the oligonucleotide administered in a particular dosage regimen. The term “therapeutically effective amount” refers to a pharmaceutical composition containing an oligonucleotide that is effective for the purpose of the present invention to achieve its intended purpose without undesirable side effects (eg, toxicity, irritation or allergic response). Refers to the quantity of an object. While individual needs may vary, determination of optimal ranges for effective amounts of pharmaceutical compositions is within the skill of the art. Human doses can be extrapolated from animal studies (Katocs et al., Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, Chapter 27). In general, the dosage required to obtain an effective amount of the pharmaceutical composition, which can be adjusted by one skilled in the art, is dependent on the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient. , The frequency of treatment, the nature of the current treatment, if any, and the nature and extent of the desired effect (s) (Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed). , Hardman et al., Eds., McGraw-Hill, New York, NY, 1996, Nies et al., Chapter 3.).
[0097]
After successful treatment, it may be desirable to subject the patient to maintenance therapy to prevent the recurrence of the treatment state, wherein the nucleic acid is administered at a maintenance dose ranging from 0.01 μg to 100 g per kg body weight, Administer at least once a day, once every 20 years. For example, in individuals known or suspected of being prone to an autoimmune or inflammatory condition, a prophylactic dose in the range of 0.01 μg to 100 g per kg body weight may be administered once or more than 20 times a day. A prophylactic effect can be achieved by administering once a year. In a similar manner, it reduces the individual's susceptibility to an inflammatory condition expected to result from certain medical treatments, such as graft-versus-host disease resulting from transplanting cells, tissues or organs into the individual be able to.
[0098]
In another method of the invention, a first antisense oligonucleotide targeted to a first LFA-3 protein is combined with a second antisense oligonucleotide targeted to a second LFA-3 protein. It is used in combination with a sense oligonucleotide to modulate such LFA-3 protein to a greater extent than can be achieved when using either oligonucleotide individually. In various aspects of the invention, the first and second LFA-3 proteins targeted by such oligonucleotides are the same, different LFA-3 proteins, or different isoforms of the same LFA-3 protein. It is.
[0099]
In some cases, it may be more effective to treat a patient with a composition comprising one or more antisense compounds of the present invention in conjunction with other traditional treatment modalities to increase the efficiency of the treatment regimen. possible. In the context of the present invention, the term "treatment regime" is intended to encompass treatment, palliative and prophylactic modes. Following treatment, the patient is monitored for changes in his / her status and for reduction of symptoms of the disorder or disease state. The dosage of the therapeutic or pharmaceutical composition can be increased if the patient does not respond significantly to the current dosage level, or if a reduction in symptoms of the disorder or disease state is observed, or The dose may be reduced if the disorder or disease state has been eliminated.
[0100]
Preventive modalities for high-risk individuals are also encompassed by the present invention. As used herein, the term “high-risk individual” means that the individual is more likely to be susceptible to the onset or recurrence of the disease or disorder than normal, eg, by individual or family history or genetic testing. Is intended to refer to the individual for which is determined. As part of a treatment regimen for a high-risk individual, the individual can be treated prophylactically to prevent the onset or recurrence of the disease or disorder. The term “prophylactically effective amount” is intended to refer to an amount of a pharmaceutical composition that produces the effect observed as preventing the onset or recurrence of a disease or disorder. A prophylactically effective amount of pharmaceutical compositions is typically determined by the effect they have compared to the effect observed when a second pharmaceutical composition lacking the active agent is administered to an individual in a similar situation. Is determined by
The therapeutic and pharmaceutical compositions of the present invention can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Typically, either oral or parenteral administration is used.
[0101]
B. Parenteral delivery: "Parenteral delivery" refers to the administration of one or more antisense compounds of the invention to an animal other than through the gut. Parenteral administration includes intravenous (iv) infusion, subcutaneous, intraperitoneal (ip) or intramuscular injection, or intrathecal or intraventricular administration. Compositions for parenteral, intrathecal or intraventricular administration include sterile aqueous solutions which may also include buffers, diluents and other suitable additives. Means for preparing and administering parenteral pharmaceutical compositions are known in the art (eg, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, 45 pg. Avis at 1569, Chapter 84). Parenteral delivery means include, but are not limited to, the following illustrative examples.
[0102]
Intravitreal injection (for delivering drugs directly to the vitreous humor of the mammalian eye) is described in US Pat. No. 5,591,720, the contents of which are incorporated herein by reference. Means for preparing and administering ophthalmic formulations are known in the art (e.g., Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, 15-95, page 15). See Mullins et al., Chapter 86).
[0103]
Intravenous administration (administration of antisense oligonucleotides to various non-human mammals) has been described by Iversen (Antisense Research and Applications, Crooke et al., Eds., CRC Press, Boca Raton, FL, FL). 1993, pages 461-469, chapter 26). Systemic delivery of oligonucleotides to non-human mammals by intraperitoneal means has also been described (Dean et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 11766).
[0104]
Diseases or conditions in which intracavitary drug administration (for administering a drug directly to an isolated portion of a tubular organ or tissue (eg, artery, vein, ureter or urethra)) affects the lumen of such organ or tissue May be desirable for the treatment of patients. To achieve this mode of oligonucleotide administration, a catheter or cannula is surgically introduced by appropriate means. For example, to treat the left common carotid artery, a cannula is inserted through the external carotid artery. After isolating the portion of the tubular organ or tissue in which treatment is sought, the composition containing the antisense compound of the present invention is injected into the isolation compartment through a cannula or catheter. Incubate for about 1 to about 120 minutes, during which time the oligonucleotide is taken up by cells in the vascular lumen, after which the tubular can be removed by removing the infusion cannula or catheter and removing the ligature that had isolated the compartments. Restoration of flow within organs or tissues (Morishita et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 8474). Antisense compounds can also be combined with a biocompatible matrix, such as a hydrogel material, and applied directly to vascular tissue in vivo (Rosenberg et al., US Pat. No. 5,593,974, issued Jan. 14, 1997). ).
[0105]
Intraventricular drug administration (for delivering the drug directly to the patient's brain) may be desirable for treating patients with diseases or conditions that affect the brain. To achieve this mode of oligonucleotide administration, a silicone catheter is surgically introduced into the ventricle of the human patient's brain, which is implanted surgically in the abdominal region (Medtronic Inc., Minneapolis, Minn.). , MN) (Zimm et al., Cancer Research, 1984, 44, 1698; Shaw, Cancer, 1993, 72 (11 Suppl., 3416)), which is used for oligonucleotide injection and an external programming device. Allows precise dosing adjustments and changes in dosing schedules with the help of. The reservoir volume of this pump is 18-20 mL and infusion rates can range from 0.1 mL / hr to 1 mL / hr. Frequency of administration ranging from to monthly Depending on the dose of the drug to be administered, ranging from 0.01 μg to 100 g per kg of body weight and body weight, the reservoir of the pump can be refilled every 3 to 10 weeks. Can be achieved by percutaneous puncture of the self-sealing septum.
[0106]
Intrathecal drug administration (to introduce the drug into the patient's spine) may be desirable for treating patients with diseases of the central nervous system (CNS). To achieve this route of oligonucleotide administration, a silicone catheter is surgically implanted into the L3-4 interlumbar space of a human patient, which is connected to a subcutaneous infusion pump that is surgically implanted in the upper abdominal region. (Luer and Hatton, The Annals of Pharmacotherapy, 1993, 27, 912, 1993; Ettinger et al. Cancer, 1978, 41, 1270; Yaida et al., Regul, 1985, 1985, 1985, Yep et al., 1985, 1985). This pump is used for infusion of oligonucleotides and allows precise dosing adjustments and changes in dosing schedules with the aid of external programming equipment. The reservoir volume of this pump is 18-20 mL, and the infusion rate can vary from 0.1 mL / hr to 1 mL / hr. Depending on the frequency of drug administration, ranging from daily to monthly, and the dose of drug to be administered, ranging from 0.01 μg to 100 g per kg body weight, pump reservoirs should be spaced every 3 to 10 weeks. Can be refilled. Refilling of the pump is achieved by a single percutaneous puncture to the self-sealing septum of the pump. The distribution, stability and pharmacokinetics of the oligonucleotide within the CNS are followed according to known methods (Whitesell et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 4665).
[0107]
To achieve delivery of oligonucleotides to areas other than the brain or spinal column by this method, the silicone catheter is configured to connect a subcutaneous infusion pump, for example, to the hepatic artery for delivery to the liver (Kemenny et al. al., Cancer, 1993, 71, 1964). Infusion pumps can also be used to achieve systemic delivery of oligonucleotides (Ewel et al., Cancer Res., 1992, 52, 3005; Rubenstein et al., J. Surg. Oncol., 1996, 62, 194).
[0108]
Epithelial and transdermal delivery (where the pharmaceutical composition containing the drug is applied topically) can be used to administer the drug to be absorbed into the local dermis or to further penetrate and absorb the underlying tissue, respectively. it can. Means of preparation and topical administration of the medicament are known in the art (eg, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, pages 160-96). Block, chapter 87).
[0109]
Vaginal delivery provides local treatment and avoids first-pass metabolism, digestion by digestive enzymes, and potential systemic side effects. This mode of administration may be preferred for antisense oligonucleotides targeted against pathogenic organisms where the vagina is a normal habitat, such as Trichomonas vaginalis. In another embodiment, an antisense oligonucleotide to a gene encoding a sperm-specific antibody can be delivered by this mode of administration to increase the likelihood of conception and subsequent pregnancy. Vaginal suppositories (Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 160-1-16, or topical delivery of Blocks 1609-1614, ointment, Chapter 87). Can be used to
[0110]
Intravesical delivery results in local treatment, avoiding first-pass metabolism, digestion by digestive enzymes, and potential systemic side effects. However, this method requires urinary catheterization of the patient and skilled staff. Nevertheless, this mode of administration may be preferred for antisense oligonucleotides targeted against pathogenic organisms that enter the urogenital tract, such as Trichomonas vaginalis.
[0111]
C. Dietary delivery: The term "dietary delivery" refers to the administration of a composition comprising one or more antisense compounds of the present invention directly or otherwise to a portion of the digestive tract of an animal. The term "gastrointestinal tract" refers to a tubular passageway in the animal running from the mouth to the anus, and any and all of its parts or segments, such as the oral cavity, esophagus, which function in digestion and absorption of food and elimination of food residues , Stomach, small and large intestine, and colon, as well as their composite parts, such as the gastrointestinal tract. Thus, the term "dietary delivery" encompasses several routes of administration including, but not limited to, oral, rectal, endoscopic and sublingual / buccal administration. Compositions for dietary delivery include sterile aqueous solutions which may also include buffers, diluents and other suitable additives. Means for preparing and administering oral pharmaceutical compositions are well-known in the art (Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, 1561, pages 61, PAGES, 961). 1633-1665, 1666-1675 and 1676-1693, respectively, Block, Chapter 87; Rudnic, Chapter 89; Porter, Chapter 90; and Longer, Chapter 91). Preferred compositions for dietary delivery of the antisense compounds of the present invention are described in Teng et al., Co-pending US patent application Ser. No. 08 / 886,829, filed Jul. 1, 1997, the entire disclosure of which is hereby incorporated by reference. Is hereby incorporated by reference.
[0112]
Buccal / sublingual administration: Delivery of a drug through the oral mucosa has several desirable features, often including a more rapid increase in drug plasma concentration than oral delivery (Remington's Pharmaceutical). Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, page 711, Harvey, chapter 35). In addition, since the venous flow from the mouth is to the superior vena cava, this route also bypasses the rapid first-pass metabolism by the liver. Both features contribute to the sublingual pathway, which is the mode of choice for nitroglycerin (Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., Eds., Ned. NY, 1996, page 7, Benet et al., Chapter 1).
[0113]
Endoscopic administration: Endoscopes can be used to deliver drugs directly to the internal parts of the digestive tract. For example, endoscopic retrograde gall bladder duct pancreatography (ERCP) utilizes an elongated gastroscope to allow selective access to the bile duct and pancreatic duct (Hirahata et al., 1988). Gan To Kagaku Ryoho, 1992, 19 (10 Suppl.), 1591). Pharmaceutical compositions containing liposome preparations can be administered by endoscopic means to parts of the gastrointestinal tract, such as the duodenum (Somogyi et al., Pharm. Res., 1995, 12, 149) or the submucosal tissue of the stomach (Akamo et al.). , Japane J. Cancer Res., 1994, 85, 652). A gastric lavage device (Inouet et al., Artif. Organs, 1997, 21, 28) and a percutaneous endoscope delivery device (Pennington et al., Alignment. Pharmacol. Ther., 1995, 9, 471) are also pharmaceutical compositions. Can be used for direct dietary delivery.
[0114]
Rectal administration: Drugs administered by the oral route can often, instead, be administered to the rectum or lower intestine by the lower intestinal route, ie, through the anus. Rectal suppositories, retention enemas or rectal catheters can be used for this purpose, and if it is otherwise difficult to achieve patient compliance (eg, in pediatric and geriatric applications, or vomiting by patients) If you are unconscious or unconscious). Rectal administration can produce higher blood levels more rapidly than the oral route, but vice versa (Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990, page 711, Harvey, chapter 35). Administration by this route greatly reduces the likelihood of first-pass metabolism, as approximately 50% of the drug absorbed from the rectum bypasses the liver (Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman. al., eds., McGraw-Hill, New York, NY, 1996, Benet et al., Chapter 1).
[0115]
Oral administration: The preferred method of administration is oral delivery, which is typically the most convenient route to access the systemic circulation. Absorption from the gastrointestinal tract is governed by factors that are generally applicable, such as surface area for absorption, blood flow to the site of absorption, the physical state of the drug and its concentration at the site of absorption (Goodman & Gilman's). The Pharmacologic Basis of Therapeutics, 9th Ed., Hardman et al., Eds., McGraw-Hill, New York, NY, 1996, pages 5-7, 1 et. Orally administered compositions containing certain oligonucleotides are known in the art (see, eg, Agrawal et al., US Pat. No. 5,591,721). Preferred compositions for oral delivery of the antisense compounds of the present invention are co-pending U.S. Patent Application No. 08 / 886,829 to Teng et al., Filed July 1, 1997, which is hereby incorporated by reference. )It is described in.
Preferred compositions for oral delivery of the antisense compounds of the present invention are co-pending U.S. Patent Application No. 08 / 886,829 to Teng et al., Filed July 1, 1997, which is hereby incorporated by reference. )It is described in.
[0116]
【Example】
The following examples illustrate the invention but are not intended to limit it. Those skilled in the art will recognize, or be able to ascertain by routine experimentation, many equivalents of the specific materials and procedures described herein. Such equivalents are considered to be within the scope of the present invention.
[0117]
Example 1: Oligonucleotide synthesis
A. General Synthesis Techniques: Oligonucleotides were synthesized on an automated DNA synthesizer using standard phosphoramidite chemistry using iodine for oxidation. β-cyanoethyldiisopropyl phosphoramidite was purchased from Applied Biosystems (Foster City, CA). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced with a 0.2 M solution of 3H-1,2-benzodithiole-3-one-1,1-dioxide in acetonitrile for stepwise thiation of the phosphite linkage. .
The synthesis of 2'-O-methyl (aka 2'-methoxy-) phosphorothioate oligonucleotides was performed using 2'-O-methyl β-cyanoethyldiisopropyl phosphoramidite (Chemgenes, Needham) instead of the standard phosphoramidite. , MA) and follow the procedure described above to increase the waiting cycle after pulse delivery of tetrazole and base to 360 seconds.
[0118]
Similarly, 2'-O-propyl (aka 2'-propoxy-) phosphorothioate oligonucleotides have been described in the U.S. application filed February 3, 1995 by slightly modifying this procedure. It is prepared according to the procedure disclosed in patent application 08 / 383,666.
The 2'-fluoro-phosphorothioate antisense compounds of the present invention are synthesized using 5'-dimethoxytrityl-3'-phosphoramidite and are described in U.S. patent application Ser. No. 08 / 383,666, filed Feb. 3, 1995. And US Pat. No. 5,459,255, issued Oct. 8, 1996. 2'-Fluoro-oligonucleotides were prepared using phosphoramidite chemistry and minor modifications of standard DNA synthesis protocols (i.e., deprotection was performed with methanolic ammonia at room temperature).
The 2'-methoxyethyl oligonucleotide was synthesized essentially according to the method of Martin et al. (Helv. Chim. Acts, 1995, 78, 486). For ease of synthesis, the 3 'nucleotide of the 2'-methoxyethyl oligonucleotide is a deoxynucleotide and 2'-O-CH₂ CH₂ OCH3-cytosine was 5-methylcytosine synthesized according to the procedure described below.
PNA antisense analogs were prepared essentially as described in U.S. Patent Nos. 5,539,082 and 5,539,083, both issued July 23, 1996.
[0119]
B. 5-Methyl-2'-methoxyethoxy-cytosine: An oligonucleotide having a 5-methyl-2'-methoxyethoxy-cytosine residue is prepared as follows.
(I) 2,2'-Anhydro [2- (β-D-arabinofuranosyl) -5-methyluridine]: 5-methyluridine (commercially available from ribosylthymine, Yamasa, Choshi, Japan) (72) 0.0g, 0.279M), diphenyl carbonate (90.0g, 0.420M) and sodium bicarbonate (2.0g, 0.024M) were added to DMF (300mL). The mixture was heated to the reflux temperature with stirring to release the released carbon dioxide gas in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethyl ether (2.5 L) with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (about 400 mL). The solution was poured into fresh ether (2.5 L) to give a hard gum. The ether was decanted and the gum was dried in a vacuum oven (60 ° C., 1 mm Hg for 24 hours) to give a solid which was crushed to a light tan powder (57 g, 85% crude yield). . This material was used as such for further reactions.
[0120]
(Ii) 2′-O-methoxyethyl-5-methyluridine: 2,2′-anhydro-5-methyluridine (195 g, 0.81 M), tris (2-methoxyethyl) borate (231 g, 0.98 M) )) And 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in an oil bath preheated at 160 ° C. After heating at 155-160 ° C. for 48 hours, the vessel was opened and the solution was evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate was evaporated. The residue (280 g) is CH₃ Dissolved in CN (600 mL) and evaporated. A silica gel column (3 kg) was charged with 0.5% Et.₃ CH containing NH₂ Cl₂ / Acetone / MeOH (20: 5: 3). CH residue₂ Cl₂ (250 mL), adsorbed on silica (150 g), and loaded on a column. The product was eluted with the packing solvent to give 160 g (63%) of the product.
[0121]
(Iii) 2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine: 2′-O-methoxyethyl-5-methyluridine (160 g, 0.506M) was simultaneously added with pyridine (250 mL). The evaporated and dried residue was dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture was stirred at room temperature for 1 hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction was stirred for another hour. Next, methanol (170 mL) was added to stop the reaction. HPLC indicated the presence of about 70% product. Evaporate the solvent, CH₃ Triturated with CN (200 mL). The residue is CHCl₃ (1.5 L) and 2 × 500 mL of saturated NaHCO 3₃ And 2 × 500 mL of saturated NaCl. Organic phase Na₂ SO₄ , Filtered and evaporated. 275 g of residue were obtained. The residue is 0.5% Et₃ Purified on a 3.5 kg silica gel column packed and eluted with EtOAc / hexane / acetone (5: 5: 1) containing NH. The pure water fraction was evaporated to give 164 g of product. About 20 g of additional product was obtained from the impure fraction, giving a total yield of 183 g (57%).
[0122]
(Iv) 3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine: 2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl Combined uridine (106 g, 0.167 M), DMF / pyridine (750 mL of a 3: 1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) and stirred at room temperature for 24 hours. . The reaction was monitored by tlc by first inactivating the tlc sample by adding MeOH. When the reaction was complete as judged by tlc, MeOH (50 mL) was added and the mixture was evaporated at 35 ° C. CHCl residue₃ (800 mL) and extracted with 2 × 200 mL of saturated sodium bicarbonate and 2 × 200 mL of saturated NaCl. Return the aqueous layer and add 200 mL CHCl₃ Extracted. The combined organics were dried over sodium sulfate and evaporated to give 122 g of residue (about 90% product). The residue was purified on a 3.5 kg silica gel column and eluted with EtOAc / hexane (4: 1). Evaporation of the pure product fractions gave 96 g (84%).
[0123]
(V) 3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine: 3'-O-acetyl-2'-O-methoxyethyl- 5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) was added to CH₃ A first solution was prepared by dissolving in CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was added to CH₃ Added to a solution of triazole (90 g, 1.3 M) in CN (1 L), cooled to −5 ° C. and stirred with an overhead stirrer for 0.5 h. POCl₃ Was added dropwise over 30 minutes to this stirred solution maintained at 0-10 ° C. and the resulting mixture was stirred for another 2 hours. The first solution was added dropwise over 45 minutes to the latter solution. The resulting reaction mixture was stored in a cold room overnight. The salt was filtered from the reaction mixture and the solution was evaporated. The residue was dissolved in EtOAc (1 L) and the insoluble solid was removed by filtration. The filtrate was washed with 1 x 300 mL of NaHCO₃ And 2 × 300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.
[0124]
(Vi) 2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine: dioxane (500 mL) and NH₄ A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in OH (30 mL) was added at room temperature for 2 minutes. Stirred for hours. The dioxane solution was evaporated and the residue azeotroped with MeOH (2 × 200 mL). The residue was dissolved in MeOH (300 mL) and transferred to a 2 liter stainless steel pressure vessel. NH₃ Gas-saturated methanol (400 mL) was added and the vessel was heated to 100 ° C. for 2 hours (thin layer chromatography, tlc showed complete conversion). The contents of the vessel were evaporated to dryness and the residue was dissolved in EtOAc (500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated, yielding 85 g (95%) of the title compound.
[0125]
(Vii) N⁴ -Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine: 2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M Was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tlc indicated that the reaction was approximately 95% complete. The solvent was evaporated and the residue was azeotroped with MeOH (200 mL). The residue is CHCl₃ (700 mL) and saturated NaHCO₃ (2 × 300 mL) and saturated NaCl (2 × 300 mL), extracted with MgSO₄ And evaporated to give a residue (96 g). The residue is separated on a 1.5 kg silica column with 0.5% Et.₃ Chromatography was performed using EtOAc / hexane (1: 1) containing NH as the elution solvent. The pure water product fractions were evaporated to give 90 g (90%) of the title compound.
[0126]
(Viii) N⁴ -Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amidite: N⁴ -Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) in CH₂ Cl₂ (1 L). Tetrazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra (isopropyl) phosphite (40.5 mL, 0.123 M) were added with stirring under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 20 hours (tic indicated that the reaction was 95% complete). The reaction mixture is washed with saturated NaHCO₃ (1 × 300 mL) and saturated NaCl (3 × 300 mL). Return the cleaning solution to CH₂ Cl₂ (300 mL), combine the extracts, and add MgSO₄ And concentrated. The resulting residue was chromatographed on a 1.5 kg silica column using EtOAc-hexane (3: 1) as the elution solvent. Pure product fractions were combined to give 90.6 g (87%) of the title compound.
[0127]
C. Purification: Cleavage from a controlled pore glass column (Applied Biosystems), deblocking in concentrated ammonium hydroxide at 55 ° C. for 18 hours, followed by precipitation twice from 0.5 M NaCl containing 2.5 volumes of ethanol. The oligonucleotide was purified. Analytical gel electrophoresis was performed with 20% acrylamide, 8 M urea, 45 mM Tris-borate buffer, pH 7.0. Oligonucleotides and their phosphorothioate analogs were determined by electrophoresis to be greater than 80% full-length material.
[0128]
Example 2: Nucleotide sequence of oligonucleotides targeted towards LFA-3
A. Oligonucleotides targeted to nucleic acids encoding human LFA-3: Table 1 shows the nucleotide sequences of a set of oligonucleotides designed to specifically hybridize to human LFA-3 mRNA and their The corresponding ISIS and SEQ ID NOs are listed. Also included are the nucleotide co-ordinates of the target gene and the target region of the gene. The nucleotide isotope is derived from GenBank accession number Y00636, locus name “HSLFA3” (see also FIG. 2 of Wallner et al., J. Exp. Med., 1987, 166, 923). Abbreviations for the genetic indicator regions are as follows: 5'-UTR, 5 'untranslated region; AUG, translation initiation region; ORE, open reading frame; stop, translation termination region; 3'-UTR, 3' untranslated region. The location of the target sequence complementary to the oligonucleotide is shown in FIG.
The nucleotides of the oligonucleotides whose sequences are presented in Table 1 are linked by phosphorothioates, with the exception of ISIS 17159, which contains both phosphorothioate and phosphodiester linkages. These oligonucleotides are gapmers having five consecutive (2'-methoxyethoxy) modified nucleotides at the 2 'position at both the 5' and 3 'ends. The internal nucleotides within these "gapmers" are unmodified at the 2 'position (2'-deoxy).
[0129]
[Table 1]

[0130]
Example 3: Assay for antisense-mediated inhibition of LFA-3 expression in HUVEC cells
A. General Technique: To assess the activity of potential human LFA-3 regulatory oligonucleotides, human umbilical vein endothelial cells (HUVECs) from Clonetics Corporation (Walkersville, MD) (alternatively, American Type Culture Collection, ATCC CRL-1730 from Rockville, MD) was grown and treated with oligonucleotides or control solutions as described below. After collection, culture extracts were prepared and examined for LFA-3 RNA and protein concentrations (eg, Northern or flow cytometry assays, respectively). In all cases, "% expression" refers to the amount of LFA-3 specific signal in oligonucleotide treated cells relative to untreated cells (or cells treated with a control solution lacking oligonucleotide), and "% inhibition" refers to the following: Calculate:
100%-% expression =% inhibition
[0131]
B. RNA assay: mRNA expression of each LFA-3 protein was determined in a "Northern" assay using nucleic acid probes capable of specifically hybridizing to them. Nucleic acid probes specific for LFA-3 are described in these examples. These probes were radiolabeled by means known in the art (eg, Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., Eds., John Wiley & Sons, New York, 1992, New York, New York, 1992). Methods in Molecular Biology, Vol. 26: Protocols for Oligonucleotide Conjugates, Agrawal, ed., N.T. Ruth, Chapter 6; and Molecular Cloning: A Laboratory M See nual, 2nd Ed., Sambrook et al., Eds., Chapter 10 of the pages 10.1-10.70). The blot was stripped to ensure equal RNA loading and to normalize the concentration of LFA-3 transcript with respect to the G3PDH signal,³²Re-search was performed using a P-labeled glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe (Clontech Laboratories, Inc., Palo Alto, Calif.).
[0132]
HUVEC (human) cells were grown in T-75 flasks to 80-90% confluence in EGM (Clonetics, Walkersville, MD) medium with 10% fetal bovine serum (FBS). At this point, cells were washed three times with 10 mL of Opti-MEM medium (GIBCO-BRL). Next, 10 μ / mL LIPOFECTIN® (ie, 1: 1 (w / w) DOTMA / DOPE, where DOTMA = N- [1- (2,3-dioleoxy) propyl] -N, N, 5 mL of Opti-MEM medium containing N-trimethylammonium chloride and containing DOPE = dioleoylphosphatidylethanolamine (GIBCO-BRL) and an appropriate amount of oligonucleotide were added to the cells (in the experiments described herein, the oligonucleotide was The nucleotide addition time is t = 0h). As a control, cells were treated with LIPOFECTIN® without oligonucleotide for the same time and under the same conditions as the oligonucleotide-treated sample. After 4 hours at 37 ° C. (t = 4 h), the medium was replaced with fresh EGM medium containing 10% FBS. Cells were allowed to recover, typically for 2 hours. Next, total cellular RNA was extracted in guanidinium and transferred to a filter by gel electrophoresis according to techniques known in the art (eg, Molecular Cloning: A Laboratory Manual, 2nd Ed., Sambrook et al., 1988). eds., pages 7.1-7.87, Chapter 7, and Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons, New York, New York, New Zealand. -30 and 4-14 to 4-29).
[0133]
Following RNA transfer, filters are combined with a probe specific for the particular LFA-3 encoding gene of interest in a hybridization buffer (QUIKHYB ™ Hybridization Solution, Stratagene, La Jolla, CA). , Typically overnight. This is followed by washing twice at room temperature (（24 ° C.) for 15 minutes with 2 × SSC, 0.1% SDS, and once at 60 ° C. for 30 minutes with 0.1 × SSC, 0.1% SDS did. Hybridizing bands were visualized by exposure to X-OMAT AR film and quantified using PHOSPHORIMAGER® essentially according to the manufacturer's instructions (Molecular Dynamics, Sunnyvale, CA). Although PHOSPHORIMAGER® or comparable instrumental quantification is the preferred means of measuring RNA concentration, the results of these "Northern" assays can be determined by other means known in the art.
[0134]
C. Protein assay: HUVEC cells were grown in 12-well plate flasks to 80-90% confluence in EGM (Clonetics, Walkersville, MD) medium with 10% FBS. At this point, cells were washed three times with 10 mL of Opti-MEM medium (GIBCO-BRL). Next, 10 μg / mL LIPOFECTIN® [ie, 1: 1 (w / w) DOTMA / DOPE, where DOTMA = N- [1- (2,3-dioleoxy) propyl] -N, N, 500 μL Opti-MEM medium containing N-trimethylammonium chloride, DOPE = dioleoylphosphatidylethanolamine; GIBCO-BRL) and an appropriate amount of oligonucleotide were added to the cells (in the experiments described here). The addition time of the oligonucleotide is t = 0h). As a control, treated with LIPOFECTIN® without oligonucleotide for the same time and under the same conditions as the oligonucleotide-treated sample. After 4 hours at 37 ° C. (t = 4 h), the medium was replaced with fresh EGM medium containing 10% FBS. Cells were typically allowed to recover for 48 hours prior to preparation of the protein extract. Cells were washed and released by treatment with trypsin (GIBCO-BRL). Cells are stained with an appropriately fluorescently labeled primary antibody that specifically recognizes the LFA-3 protein under test and is subjected to fluorescence activated cell sorting (FACS®) technology (see, eg, Recktenwald US Pat. , 727,020, Kortright et al., 5,223,398 and Sizto et al., 5,556,764) were used to determine the amount of each LFA-3 protein. Fluorescently labeled primary antibodies specific for each LFA-3 protein are described in the appropriate examples. Instead, detection is performed by using an unlabeled primary antibody against LFA-3 (some of which are described in these examples) and using a fluorescently labeled secondary (ie, specific to the primary antibody) antibody. be able to.
[0135]
In an alternative method for measuring LFA-3 concentration, cell lysates and protein extracts are electrophoresed (SDS-PAGE), transferred to nitrocellulose filters, and detected by means known in the art (eg, Molecular Cloning: See Chapter 18 of A Laboratory Manual, 2nd Ed., Sambrook et al., Eds., Pages 18.34, 18.47-18.54 and 18.60-18.75). An unlabeled primary antibody to LFA-3 is used and detected by means known in the art, including, for example, detection of the primary antibody by a secondary antibody that binds to the primary antibody (eg, Short Protocols in Molecular Biology, 2nd). Ed., Ausubel et al., Eds., John Wiley & Sons, New York, 1992, pages 10-33 to 10-35; and Molecular Cloning, A. Laboratory, Second Edition, A. Laboratory Materials. pages 18.1-18.75 and 18.86-18.88, section 18) and quantification using other antibody-based assays known in the art. It can be. Such antibody-based assays include, but are not limited to, ELISA assays, Western assays, and the like (eg, US Patent No. 4,879,219 to Wands et al. And No. 4 to Schoemaker et al.). No., 837,167 and Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., Eds., John Wiley & Sons, New York, 1992, 11-pages. The activity of LFA-3 can be measured in appropriate cell adhesion assays and model systems known in the art (Dustin et al., Annu. Rev. Immunol., 1991, 9, 27).
[0136]
Example 4: Antisense-mediated inhibition of human LFA-3 expression by oligonucleotides
A. Activity of LFA-3 Oligonucleotides: In an initial screen for active compounds capable of modulating LFA-3 expression, HUVEC cells were treated with a 10 nM series of LFA-3-specific "gapmer" oligonucleotides (ISIS No. 16371-16381). , 16909-16912, 17092 and 17159; SEQ ID NOs: 2-15), and the concentration of LFA-3 protein was converted by FACS® to phycoerythrin (PE) from Pharmingen (San Diego, CA). Bound was determined using a monoclonal antibody against LFA-3 (CD58). An isotype (mouse IgG-2a kappa) control antibody (also from Pharmingen) typically resulted in ≤5% of the basal signal. PE- and FITC-labeled antibodies to LFA-3 are available, for example, from Immunotech (Westbrook, ME), Serotec (Oxford, UK), Biodesign International (Kennebunk, ME), Research Diagnostics, Inc. (RDI, Flanders, NJ). Primary antibodies against other LFA-3 suitable for FACS® analysis in combination with labeled secondary antibodies are described by the above vendors and, for example, Endogen (formerly T Cell Diagnostics, Woburn, Mass.) And Upstate Biotechnology (Lake). Placid, NY).
[0137]
The data from the screen (Table 2) shows the following results. In this assay, oligonucleotides exhibiting activity reflected at the level of inhibition ≧ about 30% of the LFA-3 protein concentration (where “about” indicates ± 5%) include ISIS numbers 16371, 16373, 16374, 16376, 16377, 16378, 16909, 16910 and 17159 (SEQ ID NOs: 2, 4, 5, 7, 8, 9, 5, 2, and 5, respectively). Therefore, these oligonucleotides are a preferred embodiment of the present invention for regulating LFA-3 expression. Oligonucleotides that exhibit an inhibition level ≧ about 50% of LFA-3 (where “about” indicates ± 5%) in this assay include ISIS numbers 16371, 16374, 16910, and 17159 (SEQ ID NOs: 2, 5, 2 and 5) are included. Therefore, these oligonucleotides are the most preferred embodiments of the present invention for regulating LFA-3 expression.
[0138]
[Table 2]

[0139]
B. Dose response and sequence specificity: Dose response of LFA-3 protein expression in HUVEC cells 4 hours after treatment with various concentrations of ISIS Nos. 16371, 16374, 16375 and 13315 is shown in Table 3. ISIS13315 (SEQ ID NO: 16) is an antisense oligonucleotide targeting mouse ICAM-1 and was used as an unrelated sequence control. The concentration of LFA-3 protein was determined by FACS® as described above. Under these conditions, the active antisense oligonucleotides ISIS16371 and ISIS16374, when applied at a concentration of 10 nM, result in about 45% and 70% inhibition, respectively, of the LFA-3 protein concentration, where "about" is ± Indicates 5%. In contrast, the inactive antisense oligonucleotide ISIS16375 resulted in minimal inhibition, ie, ≦ 約 15% inhibition. For ISIS 16371, the degree of inhibition increased to about 65% at 30 nM. For ISIS 16374, the degree of inhibition increased to about 80% at 30 nM.
[0140]
The specificity of LFA-3 modulation by antisense oligonucleotides was confirmed using ISIS number 13315 as a control. The results show that treatment of cells with the control oligonucleotide had little effect on LFA-3 protein expression. These results are consistent with the results shown in Table 2 for the scrambled control oligonucleotide ISIS numbers 16911, 16912, 17092. These scrambled control oligonucleotides also resulted in minimal inhibition of LFA-3, i.e., ≤ about 10%, where "about" indicates ± 5%. These results indicate that the LFA-3 modulating activity of the active antisense compound is sequence specific.
[0141]
[Table 3]

[0142]
C. Confirmation of antisense mechanism: In order to confirm that the oligonucleotide identified as active acts on the antisense mechanism, the following experiment was performed to assay the concentration of LFA-3 mRNA. To prepare an LFA-3-specific nucleic acid probe, in a PCR reaction using a PRIME-A-GENE® labeling kit (Promega, Madison, WI), RNA derived from HUVEC cells and a pair of phosphodiester oligonucleotides,
5'-CAGCGTGGTCTGCCTGCTGC (SEQ ID NO: 17); and
5'-GTTATGCTGTTGTCTTCATC (SEQ ID NO: 18),
Was used as a primer to prepare a radiolabeled LFA-3 specific PCR product. Got³²The P-cytosine radiolabeled 743 bp product hybridizes to the open reading frame of human LFA-3 (SEQ ID NO: 1). However, alternatively, other PCR probes can be prepared, or one or more of the oligonucleotides of Table 1 can be detectably labeled using methods known in the art to provide an LFA-3 specific probe. Can be used as
[0143]
Northern assays were performed as described in Example 3 using the above probes. HUVEC cells were treated with 5 nM antisense oligonucleotide and RNA was collected at t = h. This experiment was performed three times and the results were quantified using PHOSPHORIMAGER® and normalized to mRNA encoding glyceraldehyde 3-phosphate dehydrogenase (G3PDH). These results (Table 4) indicate that ISIS 16374 reduced LFA-3 mRNA levels by almost 85%, whereas control (scrambled) oligonucleotides had little effect on LFA-3 mRNA levels. Show. Therefore, the compounds of the present invention exert their antisense mechanism without losing their effects.
[0144]
[Table 4]

[0145]
D. Preferred regions for antisense modulation of nucleic acids encoding human LFA-3: Antisense compounds of the invention are designed to specifically hybridize (target) to any portion of the nucleic acid encoding LFA-3. can do. However, the above results indicate that some regions of the nucleic acid encoding human LFA-3 and sequences within such regions are particularly preferred for antisense regulation of human LFA-3, and thus are designed to regulate it. This is a preferred target region for the antisense compound to be used.
[0146]
1. The AUG (start) codon region of human LFA-3 is the target region of ISIS 16371 and 16910 (SEQ ID NO: 2), which is a preferred embodiment of the present invention.
2. The ORF (reading frame) of human LFA-3 is the target region of ISIS 16373, 16374, 16909 and 17159 (SEQ ID NOs: 4 and 5), all of which are preferred embodiments of the present invention.
3. The stop codon region of human LFA-3 is the target region of ISIS 16376 and 16377 (SEQ ID NOs: 7 and 8), all of which are preferred embodiments of the present invention. As shown in FIG. 1, these antisense compounds contain base pairs 760-800 of the human LFA-3 sequence (SEQ ID NO: 1), thus this region (SEQ ID NO: 19) regulates human LFA-3. Is a preferred target region for antisense compounds designed as follows.
4. The 3'-UTR (untranslated region) of human LFA-3 is a target region of ISIS16378 (SEQ ID NO: 9), which is a preferred embodiment of the present invention.
[0147]
E. FIG. Targeting of LFA-3 to different isoforms: In addition to the transmembrane form of LFA-3 encoded by SEQ ID NO: 1 (Wallner et al., J. Exp. Med., 1987, 166, 923) Two isoforms of LFA-3 are known. Certain antisense compounds of the present invention can be directed to a particular isoform to achieve a particular effect.
"PI-linked LFA-3" (aka PI-linked CD58) is an isoform of LFA-3 that is anchored to cell membranes by covalent attachment to phosphatidylinositol (PI). PI-linked LFA-3 is displayed on the cell surface and therefore can contribute to cell: cell association, but does not cross the cell membrane and therefore probably does not signal. The nucleotide sequence of the cDNA encoding PI-linked LFA-3 (Seed, Nature, 1987, 329, 840) differs from that encoding the transmembrane isoform at positions 719-1040 (SEQ ID NO: 1). Thus, antisense compounds targeted toward this region, including but not limited to ISIS 16376, 16377 and 16378 (SEQ ID NOs: 7, 8 and 9), are responsible for the expression of PI-linked LFA-3 It is expected that the transmembrane morphology will be reduced or inhibited without significant impact.
[0148]
"Soluble LFA-3" (aka sLFA-3 or sCD58) has been identified in human serum and urine and in culture supernatants of several human cell lines (Hoffman et al., Eur. J. Immunol., 1993, 23, 3003). The in vivo mechanism by which sLFA-3 is produced has not yet been characterized, but based on similar biological systems, (1) LFA producing mRNA encoding an isoform lacking the transmembrane domain, -3 can be caused either by alternative splicing of the RNA encoding -3 or (2) proteolytic cleavage of the membrane-bound LFA-3 protein. Without wishing to be bound by any particular theory, if sLFA-3 is produced by alternative splicing, the antisense targeted to the portion of the RNA encoding the transmembrane domain of LFA-3 Compounds are expected to reduce or inhibit the expression of the transmembrane form of LFA-3 without significantly affecting the expression of sLFA-3. The transmembrane domain is encoded by bases 655-1040 of SEQ ID NO: 1 (Wallner et al., J. Exp. Med., 1987, 166, 923), an antisense compound specifically targeted to this region Include, but are not limited to, ISIS 16376, 16377, and 16378 (SEQ ID NOs: 7, 8, and 9). Such antisense compounds are expected to reduce or inhibit the expression of the transmembrane form of LFA-3 produced by alternative RNA splicing without significantly impacting the expression of soluble LFA-3.
[0149]
Example 5: Antisense-mediated inhibition of T cell stimulation
Studies have shown that human endothelial cells (ECs) have been shown to produce interleukin 2 (IL-2), interleukin 4 (IL-4) and interferon-gamma (IFN-γ) from T cells activated by phytohemagglutinin (PHA). ) Has been shown to effectively costimulate the production of Blocking experiments using a monoclonal antibody against LFA-3 suggest that LFA-3: CD-2-mediated signaling contributes to about 50% of costimulation by EC (Hughes et al., J. Exp. Med., 1990, 171, 1453). The following experiments were performed to evaluate the ability of the antisense compounds of the present invention to modulate the ability of cells to activate T cells, as measured by cytokine production and T cell proliferation.
[0150]
A. PHA co-stimulation assay: Continuously cultured HUVEC cells were previously plated in a 6-well tissue FALCON® culture plate (Becton-Dickinson and Co., Franklin Lakes, NJ) using LIPOFECTIN®. Treated with 25 nM ISIS 16374 (active LFA-3 oligonucleotide) or 17092 (16374 scrambled control) as described in the examples. Seventy hours later, samples of treated cells were analyzed for surface expression of LFA-3 by FACS® analysis, as in the previous example, using FITC-conjugated anti-CD58 (Immunotech, Westbrook, ME). Surface expression of LFA-3 on ISIS 16374-treated HUVEC cells was reduced to 20-25% of that expressed on the surface of cells treated with the scrambled control oligonucleotide ISIS 17092. Remaining cells were replated into tissue culture round bottom 96 well plates for the following costimulation assays.
[0151]
CD4⁺   When T cells were isolated from peripheral blood monocytes (PBMC) from adult volunteer donors by negative selection, the resulting cells contained approximately 90% CD4⁺ Met. About 2,000 purified CD4⁺   T cells were collected from each well in RPMI 1640 medium (Life Technologies, Inc.) containing 10% fetal calf serum (FCS), 2.5 mM glutamine, 100 U / ml penicillin and 100 μg / mL streptomycin (all from Life Technologies) in a final volume of 200 μL. Inc., Gaithersburg, MD). The T cell mitogen phytohemagglutinin (PHA-L from Sigma Chemical Co., St. Louis, MO) was also added to the medium at 3 μg / mL.
[0152]
For comparison purposes, the blocking agent IE6 (anti-CD58, isotype IgG1, a gift from Biogen, Cambridge, Mass.) Was added at 50 μg / mL; as a control, K16 / 16 (non-binding IgG1, ascites) was 1: 1000. Added by dilution. Other anti-CD58 blocking antibodies, for example, catalog number 8-4758N from Perceptive Diagnostics, Cambridge, MA, or European Collection of Animal Cell Cultures, Salisbury, U.S.A. K. No. 91060558 can be used. Twenty-four hours later, media was collected and assayed for various T cell producing cytokines using human IL-2, IFN-γ and IL-4 ELISA kits (Coulter Corp., Miami, FL).
[0153]
These results are shown in FIG. 2, where each data point represents the average of two wells. A scrambled control (SC) oligonucleotide, ISIS 17092, has no effect on the production of three cytokines ("SC / EC", open squares) and reduces the production of cytokines, a monoclonal against LFA-3 or CD2 Does not interfere with antibody potency ("SC / EC", shaded and filled squares, respectively). In contrast, active antisense oligonucleotide (ASO) ISIS 16374 was almost as effective in inhibiting cytokine production as antibodies against LFA-3 or CD2 ("ASO / EC", open squares). When ISIS16374 was combined with the antibody, no further decrease in cytokine production was seen ("ASO / EC", hatched and filled squares).
[0154]
B. Allo-reaction: According to previous studies, IFN-γ-treated cultured ECs⁺ Cells can be activated to secrete cytokines and proliferate, and monoclonal antibodies to LFA-3 have been shown to inhibit this allo-reaction (Savage et al., Transplantation, 1993, 56). , 128). Allo-reaction was started in the same manner as in the PHA co-stimulation assay, except that co-stimulation was performed on a flat bottom plate and no PHA was added. CD4⁺   Four days before the addition of T cells [d (-4)], ECs were applied at low confluence and immediately treated with 500 U / mL human IFN-γ to induce expression of class II MHC. d (-3) was treated with EC with an oligonucleotide, re-coated on d (-1), d0 was treated with 10 μg / mL mitomycin C (Sigma) for 1 hour, washed, and then washed with 3 × 10⁵ Pieces of purified CD4⁺   T cells were added.
[0155]
Media was collected from these co-cultures at d1, d2 or d3 for cytokine assays using an ELISA kit. In experiments measuring IL-2, anti-TAC was added at 10 mg / mL to prevent IL-2 uptake by cells from the medium. Anti-TAC is a monoclonal antibody that binds to the alpha chain of the IL-2 receptor and blocks it from interacting with IL-2 (gift from Tom Waldmann, commercially available from BioSource International, Camarillo, CA). Available). In addition, during the last 18-24 hours of coculture of d4, d5, d6 or d7, 1 μCi of [³ [H] thymidine was added to each well to increase the proliferation of T cells [³ H] uptake. The plates were harvested in a 96-well harvester (Tomtec Co. Ltd., Tokyo, Japan) and counted on a Microbeta scintillation counter (EC & G Wallac, Turku, Finland). The results are shown in FIGS. 3 and 4, where each data point represents the average of three replicate wells.
[0156]
As shown in FIG. 3, pre-treating the ECs with the active antisense oligonucleotide ISIS 16374 (closed squares) resulted in IL-2 and IFN- compared to the scrambled control oligonucleotide 17092 (open squares). Production of γ was inhibited. Although the effect on IFN-γ was not significant, ISIS 16374 against IL-2 maintained a level of about 50% inhibition for at least 3 days.
[0157]
The results from three independent proliferation assays are shown in FIG. The allo-response varied somewhat; the degree of inhibition of the peak proliferative response ranged from about 20% to about 50%. However, in all cases, the peak proliferative response is inhibited by EC pretreatment with ISIS 16374.
These results indicate that the antisense compounds of the invention specifically inhibit LFA-3 expression on human endothelial cells and that such treatment at least reduces the ability of such cells to activate T cells. It shows that it reduces to the same extent as a monoclonal antibody against LFA-3 or CD2.
[0158]
Example 6: Treatment method
The antisense compounds of the present invention produce immunosuppressive and anti-inflammatory effects in vivo, and those skilled in the art need such effects to provide prophylactic, palliative and therapeutic benefits to animals, including humans. Can be used to provide The antisense compounds of the present invention have also been evaluated for their ability to inhibit metastasis of cancer cells and are used to provide a prophylactic, palliative or therapeutic alleviation from hyperproliferative disease. Therapeutic methods using the antisense compounds of the present invention include, but are not limited to, the following examples.
[0159]
A. Modulation of unwanted immune responsive phenomena: The present invention provides methods for modulating immune responsive phenomena that are directly or indirectly mediated by or affected by LFA-3 in animals. Such an immune responsive phenomenon can lead to undesirable effects, such as inflammation. Methods of modulating immune responsive phenomena mediated by or affected by LFA-3 include, if necessary, one or more antisense compounds of the invention (or one or more antisense compounds) in a pharmaceutical formulation. Inflammatory agents or immunosuppressive non-antisense based agents, ie, combinations of NABA; see below). Some specific therapeutic modalities of the antisense compounds of the invention follow by way of example, but are not intended to limit the scope of the invention.
[0160]
1. LFA-3 and CD2-mediated events: The present invention provides compositions and methods for inhibiting LFA-3 expression. Since induction of subsequent molecular and / or cellular events requires the binding of LFA-3 to CD2, inhibition of LFA-3 expression would be expected to result in a reduction or inhibition of such events. A variety of in vitro and in vivo methods are available for assaying the ability of the antisense compounds of the present invention to inhibit the LFA-3: CD2 interaction (eg, Dustin et al., J. Cell Biol. , 1996, 132, 465; see Ding et al., J. Immunol., 1996, 157, 1863).
[0161]
2. T cell activation: The present invention is a method of modulating LFA-3-mediated T cell activation in an animal, comprising, if necessary, one or more antisense compounds of the present invention, or in a pharmaceutical formulation, in a pharmaceutical formulation. And administering one or more anti-inflammatory or immunosuppressive agents to an animal. A soluble form of LFA-3 that competes with cell surface LFA-3 for membrane-bound CD2 prevents T cell activation (Yamashita et al., Immunol., 1997, 92, 39). Unwanted LFA-3 mediated or related T cell activation is believed to be a key component in many damaging immune responses, including chronic liver disease including inflammation, asthma, and alcoholic cirrhosis. (Mengelsers et al., Am. J. Respir. Crit. Care Med., 1994, 149, 345; Hoffman et al., J. Hepatol., 1996, 25, 465; Santos-Perez et al.). , Immunol. Lett., 1996, 50, 179). Administration of an antisense compound of the present invention to an animal, if necessary, as part of a suitable pharmaceutical composition, may result in activation of T cells and subsequent undesired immune responsive phenomena such as inflammation and inflammatory damage. Inhibition is expected. Such treatments may include one or more anti-inflammatory agents and / or immunosuppressive NABAs and, in addition or alternatively, one targeted to, for example, CAM protein, B7 protein, or protein kinase C. It can be in combination with more than one additional antisense compound (see below). Such administration may be systemic or directly to the site (s) of inflammation and / or inflammatory damage. The antisense compounds of the present invention may be tested for their ability to modulate T cell activation and subsequent unwanted inflammation and / or inflammatory damage, for example, the assays described in Example 5 and / or other suitable Is evaluated in an in vitro or in vivo model.
[0162]
3. Allograft rejection and GVHD: The present invention is a method of avoiding allograft rejection, including the treatment or prevention of graft-versus-host disease (GVHD) in an animal, if necessary, in a pharmaceutical formulation. Also provided is a method comprising administering to an animal the above antisense compounds of the present invention, or a combination thereof with one or more anti-inflammatory or immunosuppressive agents. Inhibition of LFA-3: CD2 interaction by soluble LFA-3-IgG1 fusion protein prolongs survival of primate heart allografts and protects xenografted skin tissue in immunodeficient mouse / human chimeras (Kaplon et al., Transplantation, 1996, 61, 356; Sultan et al., Nature Biotech., 1997, 15, 759). Accordingly, administration of one or more LFA-3 modulatory antisense compounds of the present invention to an animal (if necessary, in combination with other agents and as part of a suitable pharmaceutical composition) allows for heterologous administration. Modulation of graft or allograft rejection and GVHD is expected.
[0163]
The administration of the antisense compound of the present invention in this method may be systemic, but may be directed to the region (one or more) of the directly transplanted tissue (s) or organ (s). Or administered to the tissue (s) or organ (s) prior to transplantation ex vivo. Such treatments may include one or more anti-inflammatory agents and / or immunosuppressive NABAs and, in addition or alternatively, one targeted to the CAM protein, B7 protein, or protein kinase C It can be a combination with the above additional antisense compounds (see below). The antisense compounds of the present invention are evaluated for their ability to modulate allograft rejection using one or more assays known in the art and / or one or more appropriate animal models (e.g., See, Stepkowski et al., J. Immunol., 1994, 153, 5336, and Example 21 of Bennett et al., US Pat. No. 5,514,788, which are incorporated herein by reference).
[0164]
5. Arthritis: The present invention is a method of treating various forms of arthritis in an animal, comprising one or more antisense compounds of the invention, or one or more anti-inflammatory compounds, if necessary, in a pharmaceutical formulation. Also provided is a method comprising administering an agent or combination with an immunosuppressive agent to an animal. Such administration can be systemic or to directly involved tissues, such as synovial fluid. A soluble form of LFA-3 (CD58) has been identified in the synovial fluid of patients with rheumatoid arthritis (RA) and is found at lower concentrations in RA patients compared to those found in non-RA humans ( Hoffman et al., Clin. Exp. Immunol., 1996, 104, 460; Hoffman et al., Clin. Exp. Rheumatol., 1996, 14, 23). Thus, the administration of one or more LFA-3-modulating antisense compounds of the invention to an animal (if necessary, in combination with other agents and as part of a suitable pharmaceutical composition) can result in RA Is expected to be adjusted.
[0165]
Administration of an antisense compound of the invention to treat RA may be in combination with one or more additional antisense compounds or anti-inflammatory / immunosuppressive NABA (see below). The antisense compounds of the present invention may be evaluated for their ability to modulate arthritis and the inflammatory damage resulting therefrom using one or more assays and / or one or more appropriate animal models known in the art. (See, eg, published PCT application WO 95/32285 to Benoist et al.).
[0166]
6. Intestinal Inflammatory Disorders: The present invention is a method of treating various intestinal inflammatory disorders in an animal, if necessary in a pharmaceutical formulation, one antisense compound of the present invention, or one thereof. Also provided is a method comprising administering to an animal a combination of the above anti-inflammatory or immunosuppressive agents. Such disorders include, for example, Crohn's disease (CD) and other forms of local enteritis; and various forms of colitis, including ulcerative colitis (UC) and granulomatous, ischemic, and radiation colitis (See The Merck Manual of Diagnosis and Therapy, 15th Ed., Pp. 797-806, Berkov et al., Eds., Rahay, NJ, 1987). An in vitro model of Crohn's disease has been described in which cellular aggregates similar to tissue granulomas found in Crohn's disease are formed. Blocking antibodies to LFA-3 inhibits in vitro aggregate formation in this model (Mishra et al., Gastroenterol., 1993, 104, 772). Furthermore, as in RA, soluble LFA-3 levels are reduced in patients with inflammatory bowel disease (Hoffman et al., Z. Gastroenterol., 1996, 34, 522). These findings indicate that one or more LFA-3-modulating antisense compounds of the invention may be administered to animals (if necessary, in combination with other agents and as part of a suitable pharmaceutical composition). Shows that regulation of Crohn's disease and other intestinal inflammatory disorders can be expected.
[0167]
Administration of the antisense compounds of the invention to treat such disorders may be in combination with one or more additional antisense compounds or anti-inflammatory agents and / or immunosuppressive NABA (see below). . The antisense compounds of the present invention are evaluated for their ability to modulate intestinal inflammatory disorders using one or more assays known in the art and / or one or more appropriate animal models ( See, e.g., Okayasu et al., Gastroenterol., 1990, 98, 694; Misra et al., Gastroenterol., 1993, 104, 772; and Example 20 of Bennett et al., U.S. Patent No. 5,514,788. These are incorporated herein by reference).
[0168]
7. Autoimmune diseases and disorders: The present invention relates to autoimmune thyroid disorders; autoimmune forms of arthritis; multiple sclerosis (MS); some forms of juvenile diabetes; myasthenia gravis; pemphigus vulgaris; Also provided is a method of treating various autoimmune diseases and disorders, including, but not limited to, lupus erythematosus (SLE or lupus). (For a review of autoimmune disorders, see Steinman, Sci. Amer., 1993, 269, 107). A preferred embodiment of the present invention involves the treatment or prevention of autoimmune thyroid disorders such as Graves' disease (thyroidism), Hashimoto's disease and de Kervan thyroiditis, where LFA-3 is expressed in such conditions. Is observed (Zheng et al., J. Autoimmun., 1990, 3, 727). The administration of an antisense compound of the present invention to animals, if necessary, as part of a suitable pharmaceutical composition, is expected to prevent or inhibit the development of autoimmune disease and subsequent undesired phenomena. Such treatments are targeted at one or more anti-inflammatory / immunosuppressive NABAs, and in addition or alternatively, for example, to CAM proteins, B7 proteins, or protein kinase C. It may be in combination with more than one additional antisense compound. Such administration may be systemic or directly to a particular tissue, depending on the nature of the disorder. For example, systemic administration may be more appropriate for SLE, whereas direct administration to the thyroid or adjacent tissues may be more effective in the case of Graves' disease. The antisense compounds of the present invention are evaluated for their ability to prevent or inhibit autoimmune diseases using appropriate assays and animal models known to those of skill in the art (see, eg, Burkhardt et al., Rheumatol. Int. , 1997, 17, 91).
[0169]
B. Treatment of hyperproliferative disorders: Patients with benign tumors and primary malignancies that are detected early in the developmental process often can be successfully treated by surgically removing the benign or primary tumor. it can. However, if not examined, cells from the malignant tumor will spread throughout the patient's body through the process of invasion and metastasis. Invasion refers to the ability of cancer cells to detach from the site of initial attachment and, for example, invade the underlying basement membrane. Metastasis occurs when (1) the cancer cells detach from their extracellular matrix, (2) the detached cancer cells travel to another part of the patient's body, often via the circulatory system, and (3) 3 shows a series of phenomena of attaching to the inappropriate extracellular matrix of the liposome, thereby creating foci that can result in secondary tumors. Normal cells have no ability to invade or metastasize and / or undergo apoptosis (programmed cell death) even if such a phenomenon occurs (Ruoslahti, Sci. Amer., 1996, 275, 72).
[0170]
Disseminated precancerous or cancerous cells often show ectopic expression of adhesion molecules that can facilitate step (3) of the metastatic process. Examples of such adhesion molecules include ICAM-1 and other CAMs (for review, see Tang et al., Invasion Metastasis, 1994, 14, 109). LFA-3 is also used in several hyperproliferative diseases, such as various myeloma (Cook et al., Acta Haematol., 1997, 97, 81; Tatsumi et al., Jpn. J. Cancer Res., 1996, 87). , 837), bladder tumors (Nouri et al., 1996, Urol. Int. 56, 6) and adult T-cell leukocytes (Imai et al., Int. J. Cancer, 1993, 55, 811). Thus, modulating one or more CAMs with the antisense compounds of the present invention reduces the ability of disseminated cancer cells to attach to distant and / or inappropriate matrices, thereby reducing primary tumors Metastasis can be regulated.
[0171]
Accordingly, the present invention also provides a method of modulating or preventing metastasis in an animal, the method comprising administering to the animal one or more antisense compounds of the present invention, if necessary, in a pharmaceutical formulation. . Such treatment may be in combination with one or more additional antisense compounds or NABA for anticancer chemotherapeutics (see below). The antisense compounds of the present invention are evaluated for their ability to modulate metastasis using one or more assays known in the art and / or one or more appropriate animal models (eg, Bennett et al.). U.S. Patent No. 5,514,788, Examples 16-18, which are incorporated herein by reference).
[0172]
C. Treatment of diseases caused by pathogens: Several studies have linked LFA-3 expression and / or function with enhanced virulence of human viruses. For example, LFA-3 is up-regulated in fibroblasts infected with cytomegalovirus (Grundy et al., Immunol., 1993, 78, 405), and this effect is due to treatment with antiviral NABA ganciclovia and foscarnet. Is not blocked by Craigen et al., Transplantation, 1996, 62, 1102). Thus, despite conventional antiviral treatment in transplant recipients, CMV-infected donor cells may overexpress LFA-3, which may be virtually pro-inflammatory, characterized by immunopathology or GVHD Or it may contribute to worsening allograft rejection. Such desirable treatment can be achieved by treating a recipient animal in vivo and / or a donor cell or tissue ex vivo, optionally in combination with an antiviral NABA, with an antisense compound of the present invention. Prevention or limitation of no effect is expected.
[0173]
In the case of human immunodeficiency virus (HIV), studies have shown that fixation of LFA-3 (CD58) by a soluble antibody against LFA-3 enhances HIV replication (Shattock et al., J. Infect. Dis., 1996, 174, 54). This effect may be due to a signaling mechanism, as antibodies to the LFA-3 ligand, CD2, activate transcription from the terminal repeats of HIV in T cells (Bressler et al., J. Am. Immunol., 1991, 147, 2290). Therefore, treating individuals with the antisense compounds of the present invention is expected to limit HIV growth and spread.
LFA-3 expression is increased in lesions present on the buccal mucosa of patients with oral lichen planus (OLP) and is therefore implicated in the pathogenesis of OLP (Kirby et al., Oral Dis., 1995, 1). , 193). Therefore, treating individuals with the antisense compounds of the invention is expected to limit the growth and spread of OLPs.
[0174]
D. Combination treatments and compositions
1. Combinations of antisense compounds: As described above, two or more antisense compounds can be administered simultaneously. Combination therapy also involves (1) one or more antisense compounds targeted to one or more CAMs (or a combination thereof with one or more anti-inflammatory / immunosuppressive or chemotherapeutic agents). ) Is administered for a first period of time, and then (2) one or more antisense compounds (or one or more thereof) targeted to one or more CAMs (In combination with anti-inflammatory or chemotherapeutic agents) by "switching" for a second period of time to administration of a second composition. Whether administered simultaneously or sequentially, preferred combinations of antisense compounds include molecules that mediate cell adhesion, for example: (1) LFA-3 and ICAM-1; (2) LFA-3 and VCAM-. 1; (3) LFA-3 and ELAM-1; and (4) LFA-3 and B7 proteins, such as those targeted to B7-1 or B7-2. B7-1 and LFA-3 act to activate T cells in a costimulatory manner (Parra et al., J. Immunol., 1997, 158, 637; Parra et al., Mol. Cell. Biol., 17, 1314), combination (4) is particularly preferred for regulating T cell activation. Antisense compounds targeted to the ICAM-1, VCAM-1, ELAM-1 and B7 proteins have all been described by Bennett et al. In US Pat. Nos. 5,514,788 and 5,591,623 and 1996. No. 08 / 777,266, filed Dec. 31, 2016.
[0175]
If desired, therapeutic antisense modulation of LFA-3 (or other CAM) expression can be combined with further treatment to achieve the level required to prevent or prevent the undesirable disorder or disease. . Such combinations include, for example, (a) two or more antisense compounds targeted to two or more CAMs, (b) one to a CAM, and another to a gene target. When treating animals with two or more antisense compounds, or (c) inflammation, targeted against CAMs in combination with non-antisense based anti-inflammatory or immunosuppressive agents This can be done by co-administration of the given antisense compounds. When attempting to treat an animal suffering from a hyperproliferative disease or disorder, antisense compounds targeted for CAM can be combined with non-antisense based chemotherapeutic agents. When used with an antisense compound of the present invention, such non-antisense based agents (NABA) can be combined in a simple combination (eg, administration of the NABA and the antisense compound) and sequentially (eg, the first NABA and Administering the antisense compound for a specified period of time followed by administration of a second NABA and the antisense compound), or in combination with one or more other such non-antisense based agents or physical treatments (eg, , NABA and antisense compounds or, for example, in the treatment of hyperproliferative disorders, the administration of one or more NABA and antisense compounds in combination with radiation therapy). If two (or more) antisense compounds or a combination of one or more antisense compounds and one or more NABAs are to be administered simultaneously in a treatment regimen, one of the preferred compositions comprises both ( Or all) components, especially those containing aseptically stabilized lipid vesicles. In the context of the present invention, the term "treatment regime" is intended to encompass treatment, palliative and prophylactic modes.
[0176]
For antiviral applications, the antisense compounds of the present invention can be combined with antisense compounds directed to the particular virus of interest. For example, for in vivo or ex vivo treatment of CMV-infected cells, an antisense compound can be combined with one or more antisense compounds targeted for CMV. Particularly preferred antisense compounds targeted to CMV and methods of treatment using them are described in US Pat. Nos. 5,442,049 and 5,595,978, which are incorporated herein by reference. )It is described in. Particularly preferred antisense compounds for HIV are described in U.S. Patent Nos. 5,166,195 and 5,591,623, which are also incorporated herein by reference. One or more non-antisense based antiviral agents, such as ganciclovir and foscarnet, can also be combined with one or more antisense compounds of the present invention. Other non-antisense based antiviral agents that are preferably combined with the antisense compounds of the present invention include US Pat. Nos. 5,523,389 and 5,627,185, which are incorporated herein by reference. As well as those described in published PCT patent WO 96/40164.
[0177]
2. Combination with chemotherapeutic agents: For the treatment of hyperproliferative disorders, the antisense compounds of the invention can be used in addition or alternatively in combination with non-antisense based chemotherapeutic agents. Examples of agents that can be used in combination with the antisense compounds of the present invention include daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis -Chloroethylnitrosourea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, Chlorambucil, nitrogen mustard, methylcyclohexylnitrosourea, Melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 5-fluorouracil (5-FU), 4-hydroxyperoxycyclophosphoro Amides, 5-fluorodeoxycyuridine 5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES). (In general, The Merck Manual of Diagnosis and Therapy, 15th Ed., Pp. 1206-1228, Berkov et al., Eds., Rah. y, N.J., 1987).
[0178]
3. Combinations with anti-inflammatory / immunosuppressants: Non-antisense based anti-inflammatory or immunosuppressants that can be used in combination with the antisense compounds of the invention include salicylates; indomethacin, ibuprofen, fenoprofen ( non-steroidal anti-inflammatory drugs (NSAIDs), including fenopofen), ketoprofen, naproxen, piroxicam, phenylbutazone, oxyfenbutazone, sulindac and meclofenamate; gold compounds, such as auranofin; D- Penicillamine; cyclophosphamide; methotrexate; azathioprine; colchicine; hydroxychloroquine; corticotropin; steroids and corticosteroids such as hydrocortisone, deoxyhydrocortisone, fludro. Ruchizon, prednisolone, methyl prednisolone, prednisone, triamcinolone, dexamethasone, but are betamethasone and paramethasone, but is not limited thereto. In general, The Merck Manual of Diagnosis and Therapy, 15th Ed. Pp. 1239-1267 and 2497-2506, Berkov et al. , Eds. , Rahay, N .; J. 1987.
[Brief description of the drawings]
1A and 1B show the sequence of the cDNA encoding human LFA-3 (SEQ ID NO: 1; GenBank accession number Y00636, locus name “HSLFA3”) and the positions of the antisense oligonucleotides described in the Examples. Shows the sequence. The cDNA sequence is described 5 'to 3' and has a vertical mark indicating every 10 bases; the cumulative number of bases in this sequence is shown in bold to the right of each line. The start (ATG) and stop (TGA) codons are in bold and double underlined. The nucleotide base sequences of the antisense oligonucleotides are listed 3 'to 5' to show their complementarity, and their ISIS numbers are shown in parentheses to the right of each line.
FIG. 2. PHA-activated CD4 by pretreating human endothelial cells with antisense oligonucleotide⁺ 4 shows that costimulation of cytokine production from T cells is inhibited. Symbols and abbreviations: "SC", scrambled control oligonucleotide (ISIS 17092); "ASO", active antisense oligonucleotide (ISIS 16374); open squares, no addition of monoclonal antibody; shaded squares, against LFA-3 Add monoclonal antibody; filled squares, add monoclonal antibody to CD2.
FIG. 3. Allogeneic CD4 by pretreating human endothelial cells with antisense oligonucleotide⁺ 4 shows that cytokine production by T cells is inhibited. As indicated, antibodies to the alpha chain of the IL-2 receptor (ie, "anti-TAC") were added in the experiments in the upper panel to prevent IL-2 utilization. Symbols: open squares, scrambled control oligonucleotide (ISIS 17092); filled squares, active antisense oligonucleotide (ISIS 16374).
FIG. 4. Pretreatment of human endothelial cells with antisense oligonucleotides results in allogeneic CD4⁺ 2 shows that cell growth is inhibited. Symbols: open squares, scrambled control oligonucleotide (ISIS 17092); filled squares, active antisense oligonucleotide (ISIS 16374).
[Sequence list]

Claims (13)

An antisense compound comprising about 8 to about 30 nucleotides linked by a covalent bond, comprising a nucleobase sequence capable of specifically hybridizing to a nucleic acid encoding human lymphocyte function-related antigen 3; An antisense compound that regulates the expression of said human lymphocyte function-related antigen 3, wherein the nucleobase sequence is selected from the group consisting of SEQ ID NOs: 2, 4, 5, 7, 8, and 9. A nucleic acid base sequence capable of specifically hybridizing with a region of a nucleic acid encoding human lymphocyte function-related antigen 3, wherein the region is a stop codon region and the nucleic acid base sequence is contained in SEQ ID NO: 19 The antisense compound according to claim 1, which specifically hybridizes to 2. The antisense compound according to claim 1, wherein the antisense compound regulates expression of a transmembrane isoform of human lymphocyte function-related antigen 3, but does not regulate expression of a soluble or phosphatidylinositol-linked isoform of human lymphocyte function-related antigen 3. The anti-antibody of claim 1, comprising one or more chemical modifications selected from the group consisting of one or more modified linkages, one or more modified nucleobases, and one or more sugar modifications. Sense compound. A method for blocking or limiting the interaction between lymphocyte function-related antigen 3 and CD2, wherein a cell expressing lymphocyte function-related antigen 3 is contacted with one or more antisense compounds according to claim 1. A method comprising: A pharmaceutical composition comprising one or more antisense compounds according to claim 1. 7. The pharmaceutical composition according to claim 6, which is formulated for dietary delivery. The pharmaceutical composition according to claim 6, which is formulated in a colloidal dispersion system. A pharmaceutical composition comprising:
(A) a nucleic acid sequence comprising 8 to 30 nucleotides linked by a covalent bond, comprising a nucleobase sequence capable of specifically hybridizing with a first nucleic acid encoding human lymphocyte function-related antigen 3, wherein the human lymphocyte function-associated antigen is a first antisense compound modulates the expression of the 3, antisense compounds thereof nucleobase sequence is selected from the group consisting of SEQ ID NO: 2,4,5,7,8 and 9; and (b) A nucleic acid base sequence that can specifically hybridize to a second nucleic acid encoding another human gene product than the lymphocyte function-related antigen 3, comprising 8 to 30 nucleotides linked by a covalent bond; And a pharmaceutical composition comprising a second antisense compound that regulates the expression of a human gene product other than the lymphocyte function-related antigen 3. The pharmaceutical composition according to claim 9, wherein the human gene product other than the lymphocyte function-related antigen 3 is selected from the group consisting of ICAM-1, VCAM-1, ELAM-1 and B7 protein. A pharmaceutical composition comprising:
(A) a nucleic acid sequence comprising 8 to 30 nucleotides linked by a covalent bond, comprising a nucleic acid base sequence capable of specifically hybridizing with a nucleic acid encoding human lymphocyte function-related antigen 3, wherein the human lymphocyte function-related is an antisense compound of one or more of modulating the expression of the antigen 3, antisense compounds thereof nucleobase sequence is selected from the group consisting of SEQ ID NO: 2,4,5,7,8 and 9; and (b) A pharmaceutical composition comprising one or more non-antisense based chemotherapeutic agents. A pharmaceutical composition comprising:
(A) a nucleic acid sequence comprising 8 to 30 nucleotides linked by a covalent bond, comprising a nucleic acid base sequence capable of specifically hybridizing with a nucleic acid encoding human lymphocyte function-related antigen 3, wherein the human lymphocyte function-related is an antisense compound of one or more of modulating the expression of the antigen 3, antisense compounds thereof nucleobase sequence is selected from the group consisting of SEQ ID NO: 2,4,5,7,8 and 9; and (b) Pharmaceutical compositions comprising one or more non-antisense based antivirals or antibiotics. A method of limiting, blocking or preventing the stimulation or activation of T cells by cells expressing LFA-3, wherein the isolated cells expressing LFA-3 are claimed in an effective amount of one or more types. Item 10. A method comprising contacting with the antisense compound according to Item 1.

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